81898709-Millipore-UF-Catalog.pdf

Ultrafiltration Application and Product Guide www.millipore.com

For over 50 years, Millipore has helped improve laboratory productivity and efficiency for researchers worldwide by delivering innovative products |and services backed by the highest levels of quality and expertise. expert ise. With the recent acquisitions of Upstate ®, Chemicon ® and Linco ®, we now offer a wide range of antibodies; stem cell-related products; protein immunodetection kits; and drug profiling products and services. The addition of these core products and capabilities to our existing sample preparation and laboratory water offering creates an extensive portfolio port folio of comprehensive solutions for immunodetection, cell biology biology,, and drug discovery discovery..

As part of our customer-focused approach to support and service, ser vice, Millipore sales and in-field technical teams receive ongoing training t raining in our customers’ research areas. This training allows them to provide technical t echnical assistance, validation services, equipment installation, and maintenance programs for your specific applications. We are also developing exciting new web tools to facilitate product selection select ion and on-line ordering. To experience all that Millipore has www.millipore.com. .millipore.com. to offer, visit www

For over 50 years, Millipore has helped improve laboratory productivity and efficiency for researchers worldwide by delivering innovative products |and services backed by the highest levels of quality and expertise. expert ise. With the recent acquisitions of Upstate ®, Chemicon ® and Linco ®, we now offer a wide range of antibodies; stem cell-related products; protein immunodetection kits; and drug profiling products and services. The addition of these core products and capabilities to our existing sample preparation and laboratory water offering creates an extensive portfolio port folio of comprehensive solutions for immunodetection, cell biology biology,, and drug discovery discovery..

As part of our customer-focused approach to support and service, ser vice, Millipore sales and in-field technical teams receive ongoing training t raining in our customers’ research areas. This training allows them to provide technical t echnical assistance, validation services, equipment installation, and maintenance programs for your specific applications. We are also developing exciting new web tools to facilitate product selection select ion and on-line ordering. To experience all that Millipore has www.millipore.com. .millipore.com. to offer, visit www

Table of Contents Contents OVERVIEW OF MEMBRANE FILTRATION

Removal of Unincorporated Label from Labeled Protein . . . . . . . . . . . . . . . . . . . 50

Membrane Processes . . . . . . . . . . . . . . . . . . 4

Rapid Rapid Purif Purifica ication tion of Monoclona Monoclonall Antibodi Antibodies es . . . 52

Recover y . . . . . . . . . . . . . . . . . . . . . . . . . . 6

A Simp Simple le Strat Strategy egy for for Prote Protein in Fract Fractiona ionation tion . . . . 54

Mode of Operation . . . . . . . . . . . . . . . . . . . 9

Urine Co Concentration . . . . . . . . . . . . . . . . . . . 56

Diafiltration . . . . . . . . . . . . . . . . . . . . . . . . . 10

Use of Centrifugal Filter Filter Devices as an Alternative to Stirred Cells . . . . . . . . . . . . . . . 58 58

Fractionation . . . . . . . . . . . . . . . . . . . . . . . . 11 11 MEMBRANES AND DEVICES FOR ULTRAFILTRATION

Membrane Selection . . . . . . . . . . . . . . . . . . 14 14 Device Selection . . . . . . . . . . . . . . . . . . . . . 16 Microcon ® Ce Centrifugal Filters . . . . . . . . . . . . . 17 17 Amicon® Ult Ultrra-4 a-4 Centri trifuga fugall Filte lters. . . . . . . . . . 18 18

PROTOCOLS FOR NUCLEIC ACIDS

Purif Purific icat ation ion of DNA Sequ Sequenc encin ingg Reac Reactio tions. ns. . . . 60 Conce Concentr ntrati ating ng and and Desalti Desalting ng DNA or or RNA. . . . 63 Preparing Samples for Forensics Identification Analysis . . . . . . . . . . . . . . . . . . 66

Amic Amicon on Ultr Ultraa-15 Cent Centririfu fuga gall Fil Filte ters. rs. . . . . . . . . . 20

Concentration of Genomic DNA for Forensic Analysis . . . . . . . . . . . . . . . . . . . . . 67 67

Centriprep ® Ce Centrifugal Filters. . . . . . . . . . . . . 22 22

Purification of PC PCR Products . . . . . . . . . . . . . . 68 68

Centricon ® Plus Plus--70 Cen Centrtrififug ugal al Filter lterss . . . . . . . . 23

Quantitative Recoveries of Nanogram Amounts of of Nu Nucleic Acids . . . . . . . . . . . . . . . 70 70

MultiScreen® Fi Filter Plate . . . . . . . . . . . . . . . . 24 24 Ultrafree Centrifugal Filters Filters . . . . . . . . . . . . . . 25

RNA Purification and Preparation of Fluorescent Fluorescent cDNA Probe from Human Human mRNA mRNA . . 72

Pellicon® XL Cassettes and Labscale™ TF T FF System . . . . . . . . . . . . . . . . . . 2266

Purification of In Vitro Syn Synth thes esiz ized ed mRNA mRNA . . . . . 76

®

Pellicon 2 Mini Cassettes . . . . . . . . . . . . . . . 28 28

Effect of Centrifugal Ultrafiltration Ultrafiltration on Large Fragment DNA Integ tegrity . . . . . . . . . . . . 78 78

Prep/Scale ® Spiral Spiral Wound Wound Filte Filterr Cartridg Cartridges es . . . 29

DNA DNA Ext Extrracti action on from from Agar garose Gel Gels. . . . . . . . . 80

Stirred Cells . . . . . . . . . . . . . . . . . . . . . . . . 30 30

PCR Purification . . . . . . . . . . . . . . . . . . . . . . 82 82

Ultrafiltration Discs . . . . . . . . . . . . . . . . . . . . 31 31

Enzyme Removal . . . . . . . . . . . . . . . . . . . . . 84 84

PROTOCOLS FOR PROTEINS

Concentration Concentration,, Desalting and Buffer Exchange . 34 Detergent Removal . . . . . . . . . . . . . . . . . . . 38 Two-Dimensional Electrophoresis Sample Pr Preparation . . . . . . . . . . . . . . . . . . . 40 40 Purifica Purification tion of Serum Serum Peptid Peptides es for Bioma Biomarker rker . . . 42

PROTOCOLS FOR VIRUS CONCENTRATION

Concentration of Bacteriophage Using Ultrafiltration . . . . . . . . . . . . . . . . . . . . . . . . 88 88 Concentration of Animal Viruses Using Ultrafiltration . . . . . . . . . . . . . . . . . . . . . . . . 91 APPENDIX

Rapid Antibody Antibody Concentration Concentration . . . . . . . . . . . . 45

High Throughput Applications . . . . . . . . . . . . 94 94

Affinit y Purification . . . . . . . . . . . . . . . . . . . . 46 46

Glossar y . . . . . . . . . . . . . . . . . . . . . . . . . . 95 95

S T N E T N O C F O E L B A T

1

S T N E T N O C F O E L B A T

2

Overview of Membrane Filtration

Membrane Processes . . . . . . . . . . . . . . . . . . .

4

Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . .

6

Mode of Operation . . . . . . . . . . . . . . . . . . . .

9

Diafiltration. . . . . . . . . . . . . . . . . . . . . . . . . . 10 Fractionation. . . . . . . . . . . . . . . . . . . . . . . . . 11

N O I T A R T L I F E N A R B M E M F O W E I V R E V O

3

Membrane Processes Ultrafiltration Ultrafiltration (UF) is the process of separating extremely small particles and dissolved molecules from fluids. The primary basis for separation is molecular size, although in all filtration applications, the permeability of a filter medium can be affected by the chemical, molecular or electrostatic properties of the sample. Ultrafiltration can only separate molecules which differ by at least an order of magnitude in size. Molecules of similar size can not be separated by ultrafiltration (see Figure 1). Materials ranging in size from 1K to 1000K molecular weight (MW) are retained by certain ultrafiltration membranes, while salts and water will pass through. Colloidal and particulate matter can also be retained. Ultrafiltration membranes can be used both to purify material passing through the filter and also to collect material retained by the filter. Materials significantly smaller than the pore size rating pass through the filter and can be depyrogenated, clarified and separated from high molecular weight contaminants. Materials larger than the pore size rating are retained by the filter and can be concentrated or separated from low molecular weight contaminants.

s e s s e c o r P e n a r b m e M • N O I T A R T L I F E N A R B M E M F O W E I V R E V O

Microfiltration Microfiltration (MF) is the process of removing particles or biological entities in the 0.025 µm to 10.0 µm range from fluids by passage through a microporous medium such as a membrane filter. Although micron-sized particles can be removed

Figure 1. Comparison of ultrafiltration with other commonly used membrane separation techniques Reverse Osmosis 0.0001 µm

U tra i tration

0.001 µm

0.01 µm

0.2 kDa

200 kDa

Sugars Amino Acids Nucleotides Salts

Micro i tration 0.1 µm

Oligonucleotides

C ari ication

1 µm

10 µm

100 µm

20,000 kDa

Proteins

Antibiotics

4

Ultrafiltration is typically used to separate proteins from buffer components for buffer exchange, desalting, or concentration. Ultrafilters are also ideal for removal or exchange of sugars, non-aqueous solvents, the separation of free from protein-bound ligands, the removal of materials of low molecular weight, or the rapid change of ionic and/or pH environment (see Figure 5, page 10). Depending on the protein to be retained, the most frequently used membranes have a nominal molecular weight limit (NMWL) of 3 kDa to 100 kDa. Ultrafiltration is far gentler to solutes than processes such as precipitation. UF is more efficient because it can simultaneously concentrate and desalt solutes. It does not require a phase change, which often denatures labile species, and UF can be performed either at room temperature or in a cold room.

Red Blood Cell

B. diminuta

Carbon Black Polio Virus Mammalian Virus

Yeast Mycoplasma

E. coli

Bacteria

Smallest Visible Particle Pollens

Clouds Fog Human Hair

by use of non-membrane or depth materials such as those found in fibrous media, only a membrane filter having a precisely defined pore size can ensure quantitative retention. Membrane filters can be used for final filtration or prefiltration, whereas a depth filter is generally used in clarifying applications where quantitative retention is not required or as a prefilter to prolong the life of a downstream membrane. Membrane and depth filters offer certain advantages and limitations. They can complement each other when used together in a microfiltration process system or fabricated device. The retention boundary defined by a membrane filter can also be used as an analytical tool to validate the integrity and efficiency of a system. For example, in addition to clarifying or sterilizing filtration, fluids containing bacteria can be filtered to trap the microorganisms on the membrane surface for subsequent culture and analysis. Microfiltration can also be used in sample preparation to remove intact cells and some cell debris from the lysate. Membrane pore size cut-offs used for this type of separation are typically in the range of 0.05 µm to 1.0 µm.

Reverse Osmosis Reverse osmosis (RO) separates salts and small molecules from low molecular weight solutes (typically less than 100 daltons) at relatively high pressures using membranes with NMWLs of 1 kDa or lower. RO membranes are normally rated by their retention of sodium chloride while ultrafiltration membranes are characterized according to the molecular weight of retained solutes. Millipore water purification systems employ both reverse osmosis membranes as well as ultrafiltration membranes. Reverse osmosis systems are primarily used to purify tap water to purities that exceed distilled water quality. Ultrafiltration systems ensure that ultrapure water is free from endotoxins as well as nucleases for critical biological research.

s e s s e c o r P e n a r b m e M •

Figure 2. Ultrafiltration membranes vs. traditional microporous membranes

Cross-section of ultrafiltration membrane with skin and porous substructure.

Ultrafiltration membranes generally have two distinct layers: a thin (0.1–1.5 µm), dense skin with a pore diameter of 10– 400 Å and a more porous substructure. Any species capable of passing through the pores of the skin (whose size is precisely controlled in manufacture) can therefore freely pass the membrane.

Microporous membranes are generally rigid, continuous meshes of polymeric material with defined pore sizes. They are used to retain bacteria, colloids and particulates. Species are either retained on the membrane surface or trapped in its substructure. Cross-section of traditional microporous membrane with uniform pore structure from top to bottom.


5

Recovery The ultimate aim of ultrafiltration is to maximize recovery of solutes of interest, but there are many membrane characteristics that affect that goal. Factors affecting recovery include: • Nominal molecular weight limit (NMWL)/ nucleotide cut-off (NCO) • Retention • Concentration polarization • Flux

Nominal Molecular Weight Limit

y r e v o c e R • N O I T A R T L I F E N A R B M E M F O W E I V R E V O

6

A microfiltration membrane’s pore size rating, typically given as a micron value, indicates that particles larger than the rating will be retained. Ultrafiltration membranes are rated according to the nominal molecular weight limit (NMWL), also sometimes referred to as molecular weight cut-off (MWCO). The NMWL indicates that most dissolved macromolecules with molecular weights higher than the NMWL will be retained. An ultrafiltration membrane with a stated NMWL should retain (reject) at least 90% of a globular solute of that molecular weight in daltons. However, for a wider safety margin, the selected cut-off should be well below the molecular weight of the solute to be retained. When solutes are to be exchanged, the cut-off should be substantially above that of the passing solute. A lower NMWL increases rejection but decreases the filtration rate for the same membrane material. Retention and product recovery are a function of a variety of other factors, including the molecular shape and size of the molecule; electrical characteristics; sample concentration and composition; operating conditions; and device or system configuration. Two membranes may have the same NMWL but will exhibit different retention of molecules within a relatively narrow range of sizes. In addition, slender, linear molecules (e.g., nucleic acids) may find their way through pores that will retain a globular species of the same weight. Retention can also be affected by hydration with counter-ions. Nevertheless, NMWL has proven to be an effective general indicator of membrane performance for globular proteins.

When using membrane ultrafiltration for sample concentration or desalting, care must be taken to select a membrane (or device) with a NMWL appropriate for the application. Because there are several considerations in determining whether a given solute will or will not be retained by a membrane of a specific cut-off, it is best to choose a device with cut-off at about one half of the molecular weight of the protein to be concentrated. This maximizes protein recovery and minimizes filtration time.

Nucleotide Cut-off (NCO) For most membranes, the NMWL is determined experimentally under a standard set of operating conditions. These analyses typically employ purified globular proteins to serve as markers or indicators of the retention characteristics of an ultrafiltration membrane. Although this approach is useful for choosing the appropriate NMWL for most protein research applications, selection of a membrane with an appropriate NMWL membrane for nucleic acid or polysaccharide purification is considerably more complex. By virtue of the rod-like threedimensional structures of these molecules, these types of molecules require a tighter membrane (with a smaller cut-off) than do globular proteins of the same molecular weight. It is therefore convenient to consider the membrane retention characteristics of nucleic acids as being related to their length (in nucleotides) rather than their molecular weight. Complicating matters even further are several additional factors that affect the recovery of nucleic acid fragments from a membrane of a given NMWL. These factors include: the strandedness of the DNA or RNA molecule; whether the DNA is linear, relaxed or supercoiled (for plasmid); the ionic strength of the solvent; the velocity of the process stream over the membrane; and the nature of the driving force. The overall effect is that optimal nucleic acid recovery is achieved in low salt buffers run under conditions of relatively low velocity (e.g., low vacuum pressure or low g-force). The membrane and protocol developed for the Montage® PCR centrifugal filter device takes into account these conditions in order to provide for high

recovery of small PCR products (e.g., ~150 base pairs [bp]) as quickly as possible. For purifications that are driven by vacuum, significantly tighter membranes are typically required to obtain optimal recovery. If the DNA sample is in the presence of high salt (or the device is run at a higher-than-recommended g-force), a significantly reduced DNA recovery may be observed. Under these conditions, higher DNA or RNA recovery can be achieved by using a tighter membrane. However, it will take significantly longer to complete the purification. For applications such as PCR where removal of unincorporated single-stranded primers from double-stranded DNA fragments is required, the molecular weights of the primer and DNA fragment should differ by at least an order of magnitude for efficient separation. Millipore offers devices that are specifically designed for separating and concentrating genomic DNA and PCR products by ultrafiltration.

Retention Retention, also sometimes called rejection, is a function of molecular size and shape. Nominal cut-off levels, defined with model solutes, are convenient indicators. Degree of hydration, counter ions, and steric effects can cause molecules with similar molecular weights to exhibit very different retention behavior. Many biological macromolecules tend to aggregate, or change conformation under varying conditions of pH and ionic strength, so that effective size may be much larger than the “native” molecule, causing increased rejection. Solute/ solvent and solute/solute interactions in the sample can also change effective molecular size. For example, some proteins will polymerize under certain concentration and buffer conditions while others (e.g., heme proteins) may break into corresponding subunits. Ionic interactions or π–π stacking can cause small molecules to behave similarly to molecules of greater molecular weight. When this occurs, as in the case of phosphate ions with a 500 NMWL membrane, the small molecules may not effectively permeate the membrane. Millipore recommends the selection of a membrane filter NMWL that is one half the size of the molecule of interest. Other manufacturers may recommend a smaller differential between the size

of the NMWL and the size of the molecule but Millipore’s recommendation is designed to provide maximum recovery. Please see additional information regarding membrane NMWL selection on page 14.

Concentration Polarization Another factor affecting the retention characteristics is the potential for membrane fouling, or concentration polarization. This occurs when there is an accumulation of the retained solute on the surface of the membrane. At high concentrations, a gel layer forms that can act as a secondary membrane (Figure 3). This may interfere with passage of the molecules through the membrane and can adversely affect the flow rate. In addition, pH, buffer components, and concentration can result in a protein behaving in an anomalous manner in terms of its retention or passage by UF membranes. During concentration polarization, the gel layer on the membrane surface superimposes its own rejection characteristics on those of the membrane. Usually, concentration polarization increases retention of lower-molecular weight species. A membrane with a 100K NMWL may reject 10–20% of albumin in a 0.1% solution of pure albumin. However, in the presence of larger solutes such as IgG, it may reject 90% of the albumin. Concentration polarization makes it very difficult to use UF for solute fractionation unless the solutes to be separated differ in size by at least an order of magnitude.

y r e v o c e R •

Figure 3. High concentrations


Ultrafiltration separates proteins from soluble salts. “Concentration polarization” slows down filtration. The proteins form a gel layer on the membrane surface.

7

Flux (UF Flow Rate) During ultrafiltration, it is important to balance speed with retention to obtain optimal performance. A membrane’s flux is defined as t he flow rate divided by the membrane area. Using membranes with higher NMWL ratings will increase the flow, but at the same time lower the retention. A membrane should be selected for required rejection, consistent with desired flow rate. This is determined by sur face area, macrosolute type, solubility, concentration and diffusivity, membrane type, temperature effects on viscosity and, to some extent, pressure. When concentration polarization is rate-controlling, flux is affected by solute concentration, fluid velocity, flow channel dimensions, and temperature.

Effects of Operating Parameters on Flux Pressure

y r e v o c e R • N O I T A R T L I F E N A R B M E M F O W E I V R E V O

8

When ultrafiltering dilute protein solutions or colloid suspensions, flux will increase with increasing transmembrane pressure (TMP). These effects are most apparent when operating under controlled positive pressure, such as when using a stirred cell. When the process is membrane-controlled (i.e., when the resistance of the gel layer is much smaller than that of the membrane), the flux-pressure relationship is linear. When the process is controlled by polarization (e.g., when the resistance of the gel layer is much larger than that of the membrane), flux will reach a plateau and may actually decrease with increases in pressure.

Concentration When concentration of the retained species is very low, flux is independent of concentration. As solute concentration rises during operation, increased viscosity and the polarization effect cause flux to decrease.

Temperature Increasing the operating temperature normally increases UF rates. A higher temperature increases solute diffusivity (typically 3–3.5% per degree Celsius for proteins) and decreases solution viscosity. Common practice is to operate at the highest temperature tolerated by the solutes and the equipment. An exception to the rule is fermentation broth concentration in the presence of some antifoams.

Many antifoams exhibit a phenomenon called “cloud point.” As temperatures increase, antifoam comes out of solution, forming a second phase. Increasing temperature above the cloud point causes flux to decrease.

pH Changing solution pH often changes molecular structure. This is especially true for proteins. At its isoelectric point, a protein begins to precipitate, causing a flux decrease.

Fouling Flux decrease due to concentration polarization should not be confused with the effect of membrane fouling. Fouling is usually the deposition and accumulation of submicron particles and solute on the membrane surface and/or crystallization and precipitation of smaller solutes on or within the pores of the membrane. There may be a chemical interaction with the membrane.

Importance of Recovery While rejection is used to characterize membrane performance, it does not always directly correlate with solute recover y from a sample or volume. Actual solute recovery—the amount of material recovered after ultrafiltration—is generally based on mass balance calculations. In many cases, especially when working with small samples of dilute, valuable solutions, the degree of recovery of a target solute is vitally important. In such cases, potential loss by non-specific adsorption must be considered. Different membrane materials adsorb biomolecules to varying degrees. Where maximum recovery is desired, the choice of a membrane with the least non-specific adsorbtivity is essential. Millipore’s Ultracel® regenerated cellulose membranes were specifically developed to minimize non-specific adsorption. Since adsorption is a direct function of membrane and device surface area, device size must be considered when recovery is important. Small, dilute samples should be concentrated with membranes of minimal surface area, commensurate with achievement of reasonable flow rates. Millipore offers a wide range of centrifugal devices, stirred cells, and tangential flow systems with an extensive choice of membrane areas and NMWLs.

Mode of Operation The pressure required for ultrafiltration can be supplied in a number of different ways depending on the product in use. For example, Millipore’s small volume ultrafiltration products generally use centrifugal force. Pump pressure is used with the tangential-flow filtration (TFF) products and compressed gas is utilized with the stirred cell products. In addition, Millipore provides multiwell ultrafiltration products that utilize vacuum and centrifugation.

Normal vs. Tangential Flow Filtration Filtration can be broken down into two different operational modes: normal flow filtration (NFF) and tangential flow filtration (TFF). The difference in fluid flow between these two modes is shown in Figure 4.

Figure 4. Normal flow filtration (NFF) vs. tangential flow filtration (TFF) In normal flow filtration (NFF), fluid is convected directly toward the membrane under an applied pressure. Particulates that are too large to pass through the pores of the membrane accumulate at the membrane surface or in the depth of the filtration media, while smaller molecules pass through to the downstream side. This type of process is often called dead-end filtration. However, the term “normal” indicates that Norma F ow Fi tration

Tangentia F ow Fi tration

Pressure

Feed Flow

Pressure

Feed Flow

Membrane

Membrane

Filtrate

Filtrate

the fluid flow occurs in the direction normal to the membrane surface, so NFF is a more descriptive and preferred name. NFF can be used for sterile filtration of clean streams, clarifying prefiltration, and virus/ protein separations. In tangential flow filtration (TFF), the fluid is pumped tangentially along the surface of the membrane. An applied pressure serves to force a portion of the fluid through the membrane to the filtrate side. As in NFF, particulates and macromolecules that are too large to pass through the membrane pores are retained on the upstream side. However, in this case the retained components do not build up at the surface of the membrane. Instead, they are swept along by the tangential flow. This feature of TFF makes it an ideal process for finer sizedbased separations. Although TFF is more commonly associated with large scale processing, centrifugal UF devices with vertical membrane panels, such as Amicon Ultra devices, also benefit from a TFF-like mode of separation, particularly in a swinging bucket rotor. TFF is also commonly called cross-flow filtration. However, the term “tangential” is descriptive of the direction of fluid flow relative to the membrane, so it is the preferred name.

n o i t a r e p O f o e d o M • N O I T A R T L I F E N A R B M E M F O W E I V R E V O

9

Diafiltration

n o i t a r t l i f a i D • N O I T A R T L I F E N A R B M E M F O W E I V R E V O

Millipore membranes provide an inexpensive means of separating macromolecular mixtures into sizegraded classes either by direct ultrafiltration or by diafiltration. Diafiltration removes microsolutes by adding solvent to the solution being ultrafiltered at a rate equal to the UF rate, independent of microspecies concentration. This rapid, efficient process washes microspecies from the solution at constant volume, thereby purifying the retained species. This process is most effective if the passing molecules are at least 10 times smaller than the molecules to be retained and concentrated by the membrane. Diafiltration is useful for sample desalting and buffer exchange. When diafiltration is used for sample desalting or buffer exchange, there is no resulting change in buffer composition. A solution volume with 100 mM salt still contains 100 mM salt after the initial concentration spin. Rediluting the retentate with water and spinning again effectively decreases the salt concentration of the sample by the concentration factor of the ultrafiltration. For For example, if a 4,000 µL sample containing 100 mM salt is concentrated to 50 µL (80X) in an Amicon Ultra centrifugal filter unit, rediluted with water to 4,000 µL, and reconcentrated, the salt concentration will be reduced 80X to 1.25 mM. To achieve more complete complet e salt removal, multiple concentration and redilution spins are required. For most samples, t wo concentration/reconstitution concentration/reconstitution cycles will remove remove about 99% 99% of the initial salt salt content.

Figure 5. Removing salts from retained solutes using diafiltration 100 µL UF membrane 10X concentration of protein 100 mM NaCl

10 µL

Add 90 µL Milli-Q ® water

90 µL

10-fold dilution

100 mM NaCl

The sodium chloride concentration is reduced by dilution.

10

With very small sample volumes, dilution of the sample before the initial concentration spin can often decrease salt concentration to an acceptable level. For example, if a 200 µL sample containing 100 mM salt is diluted t o 4,000 µL before concentration in an Amicon Ultra centrifugal filter unit, the salt concentration in the 4,000 µL sample will be 5 mM. The concentrate will still contain 5 mM salt. If more complete salt removal is desired, a re-dilution/spin cycle should be added. In this example, if the original spin ended with 50 µL of retentate, redilution to 4,000 µL results in 0.06 mM salt concentration. The sample can then be reconcentrated to 50 µL in an Amicon Ultra centrifugal filter device. Diafiltration can be a continuous or a discontinuous process. In continuous diafiltration, such as in a stirred cell or a TFF device, the solution is maintained at a fixed volume while solvent flows continuously through the mixture. Salts and other microsolutes are steadily removed by convective transport. Microsolute exchange can be accomplished using the same principle. Constant operator attention is not required and the possibility of solute denaturation by overconcentration is eliminated. In discontinuous diafiltration, such as in a centrifugal ultrafiltration device, salts and microsolutes are removed by repeated concentration and dilution (Figure 5).

100 µL

10 µL 10X concentration of protein

10 mM NaCl

90 µL 10 mM NaCl

Dialysis vs. Diafiltration Dialysis is a traditional method for removing microsolutes or exchanging solvents. It is a slow diffusive process generally employing regeneratedcellulose tubing as the barrier membrane. In dialysis, the process solution and exchange solvent are on opposite sides of the semi-permeable barrier membrane through which permeating microsolutes diffuse. The permeation rate of solutes from sample to dialysate is a direct ratio to the solute concentration and inversely proportional to the solutes’ molecular weight. Desalting by dialysis is timeconsuming and relatively inefficient at low concentrations. Millipore’s Millipore’s Amicon centrifugal concentrators provide a fast, convenient, high-recovery alternative to dialysis or precipitation without diluting samples. The relative merits of diafiltration and dialysis are summarized in the Table 1.

Table 1. Comparison of diafiltration and dialysis Diafiltration

Dialysis

Transport convective convec tive with solvent, independent of microsolute composition. Rapid rate. Fractional removal independent of content.

Transport diffusion-controlled, d iffusion-controlled, dependent on type of microsolute.

Ultrafiltration rate reduced with decreased temperat ure (net effect not as marked). At elevated macrosolute content, ultrafiltration rate reduced. Minimal exchange solvent required; easily contained in reservoir. Simple automation with endpoint control apparatus.

Slow transport. Lower efficiency with decreased microsolute concentration. Marked temperature dependence (reduced transport at lower temperature). Microsolute transport relatively unaffected by macrosolute content. Frequent dialysate change. Recirculation about bags to maximize transport. Automation possible with complex equipment.

Fractionation Fractionation is the process of separating a mixture into its components using a combination of physicochemical properties of the solute. Ultrafiltration membranes have been used for fractionation of protein solutions on the basis of size1. This technique is also called membrane partitioning chromatography 1,2. Here, proteins larger than the pore size are retained and those smaller than the pore size pass through into the permeate. Since fractionation is rarely absolute, acceptability of the results will depend on whether the passing solute, retained solute or both are of interest to the researcher and their levels of purity and yield. Hence fractionation efficiency is defined as a function of yield and purity relative to the starting solution3.

Factors to consider for efficient fractionation: 1. Selection of the appropriate molecular weight cutoff: Selecting the appropriate MWCO of the membrane is critical to ensure efficient fractionation. The size of the retained and the passing species affect the selection criteria for the membrane pore size5. 1. Molecular weight of of retained ≥ MWCO x 0.9 2. MWCO ≥ Molecular weight of passing x 3 3. Molecular weight of of retained ≥ Molecular weight of passing x 3

Example separation: Separating separation: Separating a 10 kDa species from a 40 kDa protein is very likely to succeed using a 30K MWCO membrane. This is due to the fact that the retained solute is >0.9X of the MWCO. Secondly the size difference between the solutes is 4X and the ratio of the MWCO to the passing solute is also 4X.

n o i t a n o i t c a r F • N O I T A R T L I F E N A R B M E M F O W E I V R E V O

11

In the case of separating an 8 kDa from a 24 kDa using a 30K MWCO, fractionation will not be successful because the 24 kDa species is only 80% of the pore size of the 30K MWCO and it may “leak” into the permeate. Thus, careful attention needs to be paid to the size of the solutes and the available MWCOs. If the application requires the retained species, then the rules are reversed. For maximal purity, the MWCO chosen should be very open, and for maximal yield, the MWCO should be very tight.

n o i t a n o i t c a r F • N O I T A R T L I F E N A R B M E M F O W E I V R E V O

2. Starting concentration of proteins: Starting concentrations of protein solutions affect fractionation efficiency 4,5. When fractionating with an Amicon Ult ra device, the st arting concentration of the passing solute, to 10 mg/ mL does not affect yields or purity of the solute in the permeate fraction. At the other end, for dilute proteins, where the concentrations are below 0.5 mg/mL, the polarizing gel-like layer does not foul the membrane. In this case we recommend using the lowest MWCO possible to prevent any trace amounts of retained solute appearing in the filtrate. Example separation: Consider separation: Consider the separation of a 17 kDa protein from a 66 kDa protein. If a MWCO of 50K is chosen (ratio of MWCO to passing ~3X), yields and purity of the passing solute are not affected even when the starting concentration of the retained is greater than 5 mg/mL. However, if the 30K MWCO is chosen to maximize purity of the passing solute, then yields are significantly affected as concentration of the retained increases to 5 mg/mL and beyond. This is due to the fact that the ratio of the MWCO to the passing solute is ~1.8X. 3. Multiple diafiltration steps to increase yields: In order to increase yields of the passing solute and to increase purity of the retained solute, multiple spins are necessary. This approach is also called as diafiltration, where the retentate fraction is diluted back to the starting volume and the sample is centrifuged again. For e.g., if 50% of the passing solute was recovered in the filtrate in the first spin, 50% of the remaining, i.e., 25% would be recovered in a subsequent subsequ ent spin. The total for 3 spins would be 50 + 25 + 12.5,

i.e. 87.5%. This relation holds true for the most part since the total amount of the retained solute is unchanged. With multiple spins, more of the passing solute goes through into the permeate, leaving the retained fraction purer and increasing the yield of the passing solute.

4. A serial fractionation strategy for compartmentalizing unknown or complex mixtures: Our recommendation for fractionation of unknown mixtures is to start by separating the proteins using the highest MWCO available. The permeate fraction from this separation is fractionated on the next highest MWCO and so on, serially. Thus, the permeate fraction from a device with a 100K MWCO membrane contains proteins <100 kDa and has undetectable amounts of proteins with higher molecular weights. Similarly, Similarly, the permeat e from the 50K MWCO contains proteins <50 kDa and so on. A point to note is that the efficiency of removing proteins higher than the cutoff depends on the size difference between the solute and the MWCO, and the starting concentration of that solute. Smaller solutes will pass through preferentially compared to the larger ones.*

References 1. Blatt Blat t WF, WF, Hudson BG, Robinson SM, Zipilivan EM. Fractionation of protein solutions by membrane partition chromatography. Nature 1967;(216)511-513. 2. Blatt WF. Membrane partition chromatography: a tool for fractionation of protein mixtures. J Agric Food Chem 1971;19:589-594. 3. Ngiam SH, Bracewell DG, Zhou Zho u Y, Y, TitchenerHooker NJ. Quantifying process tradeoffs in the operation of chromatographic sequences. Biotechnol Prog 2003;19:1315-1322. 4. Robertson BC, Zydney AL. Polarization and adsorption effects on sieving in membrane protein filtration. filt ration. ASAIO Trans Trans 1987;33: 118-122. 5. Nel RG, Oppenheim SF, Rodgers VG. Effects of solution properties on solute and permeate flux in bovine serum albumin-IgG ultrafiltration. Biotechnol Prog 1994;10:539-542. *Refer to “A “A Simple Strategy St rategy for Protein Fractionation Using Ultrafiltration” for a protocol.

12

Membranes and Devices for Ultrafiltration

Membrane Selection . . . . . . . . . . . . . . . . . . . 14 Device Selection . . . . . . . . . . . . . . . . . . . . . . 16 Microcon Centrifugal Filters . . . . . . . . . . . . . . 17 Amicon Ultra-4 Centrifugal Filters . . . . . . . . . . 18 Amicon Ultra-15 Centrifugal Filters . . . . . . . . . 20 Centriprep Centrifugal Filters . . . . . . . . . . . . . 22 Centricon Plus-70 Centrifugal Filters. . . . . . . . . 23 MultiScreen Filter Plate . . . . . . . . . . . . . . . . . . 24 Ultrafree Centrifugal Filters. . . . . . . . . . . . . . . 25 Pellicon XL Cassettes and Labscale TFF System . . . . . . . . . . . . . . . . . . . 26 Pellicon 2 Mini Cassettes . . . . . . . . . . . . . . . . 28 Prep/Scale Spiral Wound Filter Cartridges. . . . 29 Stirred Cells . . . . . . . . . . . . . . . . . . . . . . . . . 30 Ultrafiltration Discs. . . . . . . . . . . . . . . . . . . . . 31

s e s s e c o r P e n a r b m e M • N S O I E T C I A V R E T L D I F D A N R A T L S U E N R A O R F B S M E E C I M V G E D N I T D C N E A L E S S E N O A T R E B D M I E U G M

13

Membrane Selection Millipore offers a complete range of centrifugal devices used for sample concentration, purification, and desalting or buffer exchange of soluble macromolecules. Millipore products are also available for applications such as cleaning up PCR reactions, separating protein-bound from free ligands, removing restriction enzymes, and recovering oligonucleotides from agarose gels. This section provides information to aid in choosing the correct product for a particular application. Millipore offers three distinct types of membranes to choose from. This section will describe these three types of membranes and then provide information about choosing the correct membrane based on typical recoveries or application.

Types of Membranes n o i t c e l e S e n a r b m e M

Ultracel ® Ultrafiltration Membrane To concentrate or desalt dilute solutions, use Ultracel series regenerated cellulose ultrafiltration membranes. The hydrophilic, tight microstructure of Ultracel membranes assures the highest possible retention with the lowest possible adsorption of protein, DNA or other macromolecules.

• N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

14

Table 1. Membrane selection by recovery Cytochrome c (12.4 kDa)

BSA (67 kDa)

IgG (156 kDa)

3K 10K

Q

Q

Q

P

Q

Q

30K 50K

• •

Q

Q

P

Q

100K

•

•

P

NMWL

Biomax ® Ultrafiltration Membrane To concentrate or desalt higher volumes of more concentrated samples (recommended for protein concentrations greater than 1.0 mg/mL), use Biomax polyethersulfone (PES) ultrafiltration membranes. Biomax membranes are recommended for samples such as serum, plasma, or conditioned tissue culture media.

Durapore ® Microporous Membrane To clarify biological samples, recover DNA from agarose gels, retain chromatography resins or suspended solid media, use Durapore hydrophilic PVDF microporous membranes. Durapore membranes allow all soluble protein and nucleic acids to pass, retaining sub-cellular fragments, whole cell and particulate materials. Durapore membranes are extremely hydrophilic, and they provide the lowest binding of proteins and other biologicals of all commercially available microporous membranes.

Membranes Organized by Typical Recoveries For globular proteins, there is a good correlation between molecular weight and Stoke’s radius. This usually allows one to predict the recovery of a protein based on its molecular weight if a membrane with the same nominal molecular weight rating is used (see typical recovery of a panel of protein solutes in Table 1). However, in order to accommodate the wide range of potential protein solutes with different tertiary structures, we suggest initially using the “rule of two” to ensure optimal recovery.

Rule of Two For Ultracel (regenerated cellulosic) membranes, Millipore recommends using a membrane with a NMWL at least two times smaller than the molecular weight of the protein solute that one intends to concentrate.

Rule of Three For Biomax (polyethersulfone) membranes for stirred cells and TFF, Millipore recommends using a membrane with a NMWL at least three times smaller than the molecular weight of the protein solute that one intends to concentrate.

Membranes Organized by Application See Table 2 to determine which centrifugal product to use based on application and membrane.

Table 2. Membrane selection by application

Ultrafiltration Membranes

Microporous Membranes

Molecular Weight (NMWL)

Pore Size (µm)

Type*

Specialty Devices

n o i t c a r t x E l R Z E - e C ® P e G e r e l x g u p g e a a a c t o t K 5 5 n r n a m K K K 0 c r t i o K K 0 0 0 0 1 l . 2 . 4 . 6 . 0 . o i o U B 3 5 1 3 5 1 0 0 0 0 5 M M M

Protein concentration

•

Protein purification/desalting/buffer exchange Desalting of column fractions Protein isolation from cell lysates

• • •

Peptide concentration/desalting/buffer exchange Antibody concentration Virus concentration or removal Nucleic acid concentration/desalting/buffer exchange

• • • •

Oligonucleotide concentration/desalting/buffer exchange PCR cleanup Remove linkers prior to cloning

• • •

Remove labeled nucleotides Antibody purification from hybridoma cells Rapid restriction mapping Clarify samples of particulate prior to HPLC

• • •

Clarification of cell lysates and tissue homogenates Cell harvesting Natural product screening Restriction enzyme removal Bound vs. free drugs from serum/plasma (protein removal) DNA/RNA recovery from polyacrylamide gel DNA recovery from agarose gel Oligonucleotide recovery from polyacrylamide gel Removal of unincorporated label (e.g., fluorescein) from protein Removal of imidazole from His-tag fusion protein

•

•

•

•

•

•

•

• •

• •

• • •

• • •

• •

• •

•

• • •

• • •

• •

•

• •

•

•

•

•

•

•

• •

• •

• •

•

•

•

•

•

•

•

• •

• •

•

• • •

•

n o i t c e l e S e n a r b m e M

•

•

•

•

•

•

• •

• •

• •

•

•

• •

• •

• • •

• •

• •

• •

• •

• •

•

• •

N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

*Selection of ultrafiltration membrane: Ultracel regenerated cellulose membrane is ideal for protein samples and nucleic acids. Biomax polysulfone membrane is ideal for complex samples (e.g., serum).

15

Device Selection See Table 3 to determine which centrifugal product to use for protein concentration based on initial sample molecular weight and volume.

Table 3. Protein concentration devices by filtration capacity Membrane Type

Millipore Device

Small Volume Filtration Devices

n o i t c e l e S e c i v e D • N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

16

Microcon Centrifugal Filters* Ultrafree-MC Centrifugal Filters MultiScreen Filter Plate with Ultracel Membrane** Medium Volume Filtration Devices Ultrafree-CL Centrifugal Filters Amicon Ultra-4 Centrifugal Filters Amicon Ultra-15 Centrifugal Filters Centriprep Centrifugal Filters Large Volume Filtration Devices Centricon Plus-70 Centrifugal Filters Amicon Stirred Cells Series 8000, High Output, Solvent-Resistant Stirred Cells Tangential Flow Filtration (TFF) Pellicon XL 50 Ultrafiltration Devices Prep/Scale® Spiral Wound UF Modules Pellicon 2 Ultrafiltration Modules U = Ultrafiltration

M = Microporous

*Bold type indicates recommended devices. **96-well plate. Volume per well.

U M U M U U U U U U U U

0.5 mL

2 mL

4 mL

Filtration Capacity 15 20 70 1 mL mL mL L

2 L

10 L

≥10

L

Microcon Centrifugal Filters Microcon centrifugal filters are the lab standard for small volume concentration. They allow you to process macromolecular solutions up to 500 µL using any centrifuge that can accept 1.5 mL tubes. The device’s low-adsorption Ultracel YM membrane and patented invert recovery spin combine to yield unusually high recovery rates—typically >95% of the sample, with concentration factors as high as 100X.

L M 5 . 0 ≤

Highest Recovery

• Maximum starting volume: 500 µL • Typical sample concentration volume: 5–15 µL • Low-binding Ultracel-YM regenerated cellulose membrane • Solute recoveries typically >95%

Typical Protein Recovery Retentate Recovery (%) by Nominal MW Solute (Concentration)

MW

3,000

10,000

30,000

50,000

100,000

Cytochrome c (0.25 mg/mL)

12,400

94

95

—

—

—

∂-Chymotrypsinogen (1 mg/mL) Ovalbumin (1 mg/mL) Bovine Serum Albumin (1 mg/mL)

25,000 45,000 67,000

— — —

95 — —

— 95 95

— — 95

— — —

Bovine IgG Fraction II (1 mg/mL)

156,000

—

—

—

95

95

Ordering Information

Protocol Add Sample

Description

NMWL

Qty/Pk*

Microcon Filter Units

3,000

24 100 24 100 24 100 24 100 24 100

10,000 30,000 Invert into Receiver

50,000 100,000

Catalogue No.

42403 42404 42406 42407 42409 42410 42415 42416 42412 42413

*Additional package sizes available. Contact Millipore.

Recover Sample

s r e t l i F l a g u f i r t n e C n o c o r c i M • N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

For additional information, visit www.millipore.com/microcon 17

L M 4 – 1

Amicon Ultra-4 Centrifugal Filters Amicon Ultra-4 Centrifugal Filters set the performance standard for concentrating small-volume samples. Ultracel regenerated cellulose low-binding ultrafiltration membrane combines with a vertical housing for fast sample processing with high recovery. • Processes 2–4 mL in as few as 10 minutes • >90% typical sample recoveries

Ultra Recovery, Ultra Speed

• Compatible with most rotor types • Double membrane panels increase surface area and reduce filtration times s r e t l i F l a g u f i r t n e C 4 a r t l U n o c i m A • N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

• Deadstop prevents sample from spinning to dryness and eliminates the need for an inverse spin •

-marked for in vitro diagnostic use (10K NMWL)

Spin conditions: 4000 x g, swinging bucket rotor at 25 °C, 4 mL sample. 3K and 10K: Cytochrome c, 0.25 mg/mL; 30K and 50K: BSA, 1 mg/mL; 100K: lgG, 1 mg/mL.

100

) 80 % ( y r 60 e v o c 40 e R

4.0 ) L m ( e m u l o V e t a r t l i F

3.5

20

3.0 2.5

0

2.0 1.5 1.0 0.5 0 0

Brand V

3,000 NMWL 10,000 NMWL 30,000 NMWL 50,000 NMWL 100,000 NMWL 5

10

15

20

25

Spin Time (min)

Complex sample volumes of 4 mL can be concentrated or diafiltered in as few as 15 minutes.

*Brand PG has a 9K membrane.

18

Comparative Performance

Typical Spin Times

Brand PG

Brand P

Amicon Ultra-4

Devices with 10 kDa* NMWL using Cytochrome c (0.025 mg/mL). 30


MW

3,000

10,000

30,000

50,000

100,000


12,400

94

95

—

—

—

Bovine Serum Albumin (1 mg/mL) IgG (1 mg/mL)

67,000 156,000

— —

— —

95 —

94 —

— 91

L M 4 – 1

Typical recoveries for 4 mL starting volume in fixed angle rotor at 7500 x g at 25 °C. Spin times: 5K (20 minutes); 10 and 30K (10 minutes); 50K (5 minutes); 100K (15 minutes).


Protocol

Add Sample

Description

NMWL

Qty/Pk*

Catalogue No.

Amicon Ultra-4 Centrifugal Filters are assembled with centrifuge tubes and caps

3,000

8 24 96 8 24 96

UFC8 003 08 UFC8 003 24 UFC8 003 96 UFC8 010 08 UFC8 010 24 UFC8 010 96

30,000

8 24 96

UFC8 030 08 UFC8 030 24 UFC8 030 96

50,000

8 24 96 8 24 96


10,000

100,000


s r e t l i F l a g u f i r t n e C 4 a r t l U n o c i m A • N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

Recover Purified Sample

For additional information, visit www.millipore.com/ultra4 19

L M 5 1 ≤

Amicon Ultra-15 Centrifugal Filters Amicon Ultra-15 Centrifugal Filters are the premier devices for concentrating mid-volume samples. They incorporate low-binding Ultracel regenerated cellulose ultrafiltration membrane and a vertical design for maximum recovery and minimal spin times.

Ultra Recovery, Ultra Speed

• Processes up to 15 mL in as few as 10 minutes • >90% typical sample recoveries • Compatible with most rotor types • Double membrane panels increase surface area and reduce filtration times s r e t l i F l a g u f i r t n e C 5 1 a r t l U n o c i m A • N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

• Deadstop prevents sample from spinning to dryness and eliminates the need for an inverse spin •

-marked for in vitro diagnostic use (10K NMWL)

Spin conditions: 4000 x g, swinging bucket rotor at 25 °C, 15 mL sample. 3K and 10K: Cytochrome c, 0.25 mg/mL; 30K and 50K: BSA, 1 mg/mL; 100K: lgG, 1 mg/mL. 16 ) L 14 m ( e 12 m u10 l o V 8 e t 6 a r t l i 4 F

80

) % ( r 60 e v o c e 40 R

0

3,000 NMWL 10,000 NMWL 30,000 NMWL 50,000 NMWL 100,000 NMWL

2 0 0

100

20

5 10 15 20 25 30 35 40 45 50 55 60 65

Spin Time (min)

Complex sample volumes of 15 mL can be concentrated or diafiltered in as few as 15 minutes.

*Brand PG has a 9K membrane.

20

Comparative Performance

Typical Spin Times

Brand V

Brand PG

Brand P Amicon Ultra-15

Devices with 10 kDa* NMWL using Cytochrome c (0.025 mg/mL).


MW

3,000

10,000

30,000

50,000

100,000


12,400

91

93

—

—

—

Bovine Serum Albumin (1 mg/mL) IgG (1 mg/mL)

67,000 156,000

— —

— —

98 —

93 —

— 89

L M 5 1 ≤

Typical recoveries for 15 mL starting volume in swinging bucket rotor at 400 x g at 25 °C. Spin times: 5K (45 minutes); 10 and 100K (20 minutes); 30K (10 minutes); 50K (15 minutes).


Protocol

Add Sample

Description

NMWL

Qty/Pk*

Catalogue No.

Amicon Ultra-15 Centrifugal Filters are assembled with centrifuge tubes and caps

3,000

8 24 96 8 24 96


30,000

8 24 96

UFC9 030 08 UFC9 030 24 UFC9 030 96

50,000

8 24 96 8 24 96


10,000

100,000



s r e t l i F l a g u f i r t n e C 5 1 a r t l U n o c i m A • N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

For additional information, visit www.millipore.com/ultra15 21

L M 5 1 – 5

Centriprep Centrifugal Filters Use Centriprep Centrifugal Filters to concentrate and desalt high solute biological samples in the 5–15 mL volume range. The devices are compatible with most centrifuges that accommodate 50 mL centrifuge tubes.

For High Solute Samples

• Unique inverse flow mode of operation with large deadstop • Starting volume from 5–15 mL • Low-binding Ultracel regenerated cellulose membrane • Fast sample processing with typical recovery of >90% s r e t l i F l a g u f i r t n e C p e r p i r t n e C • N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

Typical Protein Recovery Retentate Recovery (%) Solute (Concentration)

Cytochrome c (0.25 mg/mL) ∂-Chymotrypsinogen (1 mg/mL) Ovalbumin (1 mg/mL) Bovine Serum Albumin (1 mg/mL) Bovine IgG Fraction II (1 mg/mL)

3,000

10,000

30,000

50,000

12,400 25,000 45,000 67,000 156,000

90 — — — —

85 90 — — —

— — 90 90 —

— — — — 90


Protocol

Description

Disassemble Device and Add Sample Reassemble and Place in Centriprep Centrifuge Until Equilibrium is Reached Decant Filtrate and Centrifuge Again Disassemble and Recover Retentate

22

Nominal MW

Centriprep Centrifugal Filters

Membrane

NMWL

Qty/Pk*

3,000

3,000

24 96

4302 4303

10,000

10,000

24 96

4304 4305

30,000

30,000

24 96

4306 4307

50,000

50,000

24 96

4310 4311


Catalogue No.

Centricon Plus-70 Centrifugal Filters The Centricon Plus-70 device can concentrate most 70 mL solutions down to 350 µL in less than 25 minutes, making it a convenient alternative to stirred cells. Typical sample recoveries are >90% with minimal sample loss due to non-specific binding.

L M 0 7 – 0 3

Convenient Alternative to Stirred Cells

• High concentration factors, with samples typically concentrated in the 50X to 200X range • Low hold-up volume • Invert spin method of recovery • Low binding Ultracel regenerated cellulose membrane • Low-binding polypropylene housing

Typical Protein Recovery Retentate Recovery (%) Solute (Concentration)

Cytochrome c (0.25 mg/mL) Bovine Serum Albumin (1 mg/mL) IgG Fraction II (1 mg/mL)

Nominal MW

3,000

10,000

30,000

50,000

12,400 67,000 156,000

— 85 —

94 95 94

— 96 91

— 84 91

Typical Spin Times

Protocol

80

Add Sample

Invert into Receiver


•

) L m ( e 60 m u l o V 40 e t a r t l i 20 F

0 0

s r e t l i F l a g u f i r t n e C 0 7 s u l P n o c i r t n e C

PBS 0.25 mg/mL BSA 1.0 mg/mL BSA 5.0 mg/mL BSA

10

20

30

40

Time (min)

5K NMWL Biomax membrane spun at 3500 x g in a swinging bucket rotor.

Ordering Information Description

Membrane

Centricon Plus-70 Centrifugal Filters

Biomax Ultracel

NMWL

Qty/Pk*

Catalogue No.

5,000 10,000 30,000 100,000

8 8 8 8

UFC7 005 08 UFC7 010 08 UFC7 030 08 UFC7 100 08



For additional information, visit www.millipore.com/centricon70 23

L M 5 . 0

<

MultiScreen Filter Plate with Ultracel-10 Membrane The first automation high throughput ultrafiltration plate for protein purification. 10,000 NMWL Ultracel regenerated cellulose membrane provides low non-specific binding and high protein recovery. • Processes from 50 to 500 µL

e n a r b m e M 0 1 l e c a r t l U h t i w e t a l P r e t l i F n e e r c S i t l u M • N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

• 95% typical retention of Cytochrome c • Compatible with standard microtiter receiver plates (300 µL, 700 µL deep well, 150 µL conical bottom) • Fast, easy centrifugal protocol • Ideally suited for instrumentation and liquid handling equipment • Uniform performance from well to well

Typical Spin Times 300

Typical Protein Recovery Membrane NMWL

Protein Solute

Typical Protein Retention (%)

10,000

Cytochrome c, 12,400 daltons (1 mg/mL)

95

10,000

BSA, 67,000 daltons (1 mg/mL)

99

300 µL sample spun at 2000 x g in swinging bucket rotor.

) 250 L µ ( e 200 m u o 150 V e t a 100 r t i F 50

0 0

10

20

30

40

50

60

Spin Time (min)

300 µL sample spun at 2000 x g in swinging bucket rotor.


MultiScreen Filter Plate with Ultracel-10 membrane

Qty/Pk*

10

Catalogue No.

MAUF 010 10


For additional information, visit www.millipore.com/ultracel 24

Buffer Cytochrome c (1 mg/mL BSA (1 mg/mL) Fetal Bovine Serum

Ultrafree Centrifugal Filters for Sample Clarification

L M 2 – 1 . 0

Ultrafree-MC Centrifugal Filters For sample clarification with low hold-up • Maximum starting volume: 500 µL • Hold-up volume: <5 µL

Ultrafree-CL Centrifugal Filters For sample clarification with high recovery • Maximum starting volume: 2 mL • Hold-up volume: <10 µL

Ultrafree-MC


Membrane

Ultrafree-MC Centrifugal Filters

Durapore (PVDF)

Pore Size (µm)

Sterility

Qty/Pk

Catalogue No.

0.1

Non-sterile

25 100

UFC3 0VV 25 UFC3 0VV 00

0.22

Non-sterile

25 100 50

UFC3 0GV 25 UFC3 0GV 0S UFC3 0GV 05 UFC3 0HV 25 UFC3 0HV 00 UFC3 0HV 0S UFC3 0DV 25 UFC3 0DV 00 UFC3 0DV 0S UFC3 0SV 00 UFC3 0LG 25 UFC3 0LH 25 UFC4 0VV 25 UFC4 0VV 00 UFC4 0GV 25 UFC4 0GV 00 UFC4 0HV 25 UFC4 0HV 00 UFC4 0DV 25 UFC4 0SV 25 UFC4 0LG 25 UFC4 0LH 25

Sterile 0.45

5.0 0.22 0.45 0.1

Sterile Non-sterile Non-sterile Non-sterile Non-sterile

0.22

Non-sterile

0.45

Non-sterile

0.65 5.0

Non-sterile Non-sterile

25 100 50 25 100 50 100 25 25 25 100 25 100 25 100 25 25

0.22 0.45

Non-sterile Non-sterile

25 25

0.65

Ultrafree-CL

Hydrophilic PTFE

Protocol

Add Sample

Ultrafree-CL Centrifugal Filters

Durapore (PVDF)

Close Lid

Hydrophilic PTFE

Additional package sizes available. Contact Millipore.

Particulates

Non-sterile Non-sterile Sterile Non-sterile

s r e t l i F l a g u f i r t n e C e e r f a r t l U • N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

Collect Sample

Filtrate

25

L 2 – 1

Pellicon XL Cassettes and Labscale TFF System Pellicon XL cassettes allow you to easily concentrate or diafilter samples using tangential flow filtration. The cassettes are available with either microfiltration or ultrafiltration membranes.

m e t s y S F F T e l a c s b a L d n a s e t t e s s a C L X n o c i l l e P • N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

26

• Use cassettes cassett es with Ultracel Ultracel regenerated cellulose or Biomax polyethersulfone (ultrafiltration) membranes for concentrating, desalting proteins, polysaccharides, lipid solutions, cell suspensions, and mammalian cells. • Use cassettes cassett es with Durapore PVDF (microporous) membrane for sample preparation, washing, or cell har vesting, or for clarif ying cell cultures, lysates, or fermentation broths.

The Labscale TFF system is specifically designed to operate Pellicon XL modules. The system reservoir accepts direct docking of the device, eliminating the need for tubing connections. • Provides the lowest working volumes, with typical sample concentrations of >25X • An optional 100 mL reservoir is available for low volume applications

Typical Processing Times Pellicon XL 150

) 125 h m L 100 ( x u l F s s e c o r P

75 50 25 0 0

1

10

100

Protein Concentration (g/L) Pellicon XL cassettes provide rapid, efficient concentration of protein-containing solutions. Data show concentration of BSA with a 10 K NMWL cassette with Ultracel regenerated cellulose membrane on a LabScale TFF system operating at 13 °C with 40 psi inlet pressure and 20 psi outlet pressure.

L 2 – 1


Membrane

Pellicon XL Cassettes

Ultracel Regenerated Cellulose

5,000 10,000 30,000 300,000 1,000,000

PXC0 05C 50 PXC0 10C 50 PXC0 30C 50 PXC3 00C 50 PXC0 1MC 50

Biomax Polyethersulfone

5,000 8,000 10,000 30,000 50,000 100,000 300,000 500,000 1,000,000

PXB0 05A 50 PXB0 08A 50 PXB0 10A 50 PXB0 30A 50 PXB0 50A 50 PXB1 00C 50 PXB3 00C 50 PXB5 00C 50 PXB0 1MC 50

Pore Size (µm)

Catalogue No.

0.10 0.22 0.45 0.65

PXVV PPC 50 PXGV PPC 50 PXHV MPC 50 PXDV PPC 50

Pore Size (µm)

Catalogue No.

Description

Membrane

Pellicon XL Cassettes

Durapore PVDF

Description

NMWL

Catalogue No.

System kit includes pump module, stir base, 500 mL and 100 mL reservoirs, sta nd for 100 mL reservoir reser voir,, and multi-manifold. Labscale TFF System

115 V 230 V GB-230 V

XX42 LSS 11 XX42 LSS 12 XX42 LSS 13

Multi-Manifold accessory allows up to three cassettes to be mounted on the Labscale system for processing multi-liter samples.

m e t s y S F F T e l a c s b a L d n a s e t t e s s a C L X n o c i l l e P • N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

27

L 0 1

Pellicon 2 Mini-Cassettes

≥

Cassette-style Pellicon 2 modules are available with Durapore PVDF (mic roporous) and Ult racel regenerated cellulose and Biomax polyethersulfone (ultrafiltration) membranes for high performance in large volume concentration and diafiltration applications, as well as scale-up applications. • Cassettes Cassett es for low viscosity (A-screen), low to medium viscosity (C-screen), and high viscosity/ high concentration (V-screen) solutions. • Larger Pellicon Pellicon cassette systems are available for processing 250 L or more. Contact Millipore or visit our web site for more information. • Stainless steel Pellicon Pellicon 2 Mini-Cassett e Holder Holder can operate up to three mini-cassettes. s e t t e s s a C i n i M 2 n o c i l l e P


Membrane

NMWL

A Screen

C Screen

V Screen

Pellicon 2 Mini-Cassettes Mini-Cassett es

Biomax Polyethersulfone

5,000 8,000 10,000 30,000 50,000 100,000 300,000 500,000 1,000,000

P2B0 05A 01 P2B0 08A 01 P2B0 10A 01 P2B0 30A 01 P2B0 50A 01 P2B1 00A 01 — — —

— — — — P2B0 50C 01 P2B1 00C 01 P2B3 00C 01 P2B5 00C 01 P2B0 1MC 01

P2B0 05V 01 P2B0 08V 01 P2B0 10V 01 P2B0 30V 01 P2B0 50V 01 P2B1 00V 01 P2B3 00V 01 P2B5 00V 01 P2B0 1MV 01

Ultracel Regenerated Cellulose

5,000 10,000 30,000 100,000 300,000 1,000,000

— — — — — —

P2C0 05C 01 P2C0 10C 01 P2C0 30C 01 P2C1 00C 01 P2C3 00C 01 P2C0 1MC 01

P2C0 05V 01 P2C0 10V 01 P2C0 30V 01 P2C1 00V 01 P2C3 00V 01 P2C0 1MV 01

• N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

28

Accessories Description

Catalogue No.

Pellicon 2 Mini Holder

XX42 PMI NI

Pellicon 2 Mini Holder Fitting Kit

XX42 PFK 01

Prep/Scale Spiral Wound Filter Cartridges Prep/Scale spiral wound filter cartridges are available in three sizes for easy, reliable preparation of samples ranging from 100 liters down to 100 mL. • Self-contained design ensures leak-free filtration • Hose barb connectors for easy setup • Can be operated with standard 1 to 16 L/minutes peristaltic pumps • Prep/Scale Holder includes fittings and basic instrumentation

s e g d i r t r a C r e t l i F d n u o W l a r i p S e l a c S / p e r P

Three Cartridge Sizes

Filter Area (m2) Minimum Working Volumes (mL)*

Prep/ScaleTFF-1

Prep/ScaleTFF-2

Prep/ScaleTFF-6

0.1 100

0.23 150

0.54 250

*Including pump tubing


Membrane

Prep/Scale Spiral Wound Filer Cartridges

Regenerated Cellulose

Polyethersulfore

NMWL

Prep/Scale-TFF-1

Prep/Scale-TFF-2

Prep/Scale-TFF-6

•

1,000 3,000 5,000 10,000 30,000 100,000 300,000 10,000 30,000 50,000 100,000 300,000

CDUF 001 LA CDUF 001 LB CDUF 001 LC CDUF 001 LG CDUF 001 LT CDUF 001 LH CDUF 001 LM CDUF 001 TG CDUF 001 TT CDUF 001 TQ CDUF 001 TH CDUF 001 TM




Accessories Description

Catalogue No.

Prep/Scale Module Holder XX42 PS0 01 Peristaltic Pumps for TFF-1 use XX8200 for TFF-2 and TFF-6 use XX80 EL

29

Stirred Cells Concentrate, diafilter, and exchange buffers for macromolecule solutions including proteins, enzymes, antibodies and viruses.

Series 8000 Stirred Cells • Five different sizes handle volumes from 3 mL to 400 mL • High flow rates with solutions up to 10% macrosolute concentration

Solvent-Resistant Stirred Cells • Two sizes: 75 mL (47 mm disc) and 300 mL (76 mm disc) Series 8000 Stirred Cell

• Borosilicate glass cylinder and PTFE components for broad chemical compatibility s e s s e c o s l r l P e C e n d a e r r b r i m t S e M • • N S O I E T C I A V R E T L D I F D A N R A T L S U E N R A O R F B S M E E C I M V G E D N I T D C N E A L E S S E N O A T R E B D M I E U G M

30

Series 8000 Models Model 8003

Model 8010

Model 8050

Model 8200

Model 8400

Maximum Process Volume (mL) Minimum Process Volume (mL)

3 0.075

10 1.0

50 2.5

200 5.0

400 10.0

Membrane Diameter (mm) Effective Membrane Area (cm2) Hold-up Volume (mL)

25 0.9 0.07

25 4.1 0.2

44.5 13.4 0.5

63.5 28.7 1.2

76 41.8 1.5


Series 8000 Stirred Cells

Solvent-Resistant Stirred Cells Solvent-Resistant Stirred Cell

Catalogue No.

Model 8003 Model 8010 Model 8050 Model 8200 Model 8400 For 47 mm membranes For 76 mm membranes

5125 5121 5122 5123 5124 XFUF 047 01 XFUF 076 01

Ultrafiltration Discs High Recovery Ultracel Ultraltration Membranes • Hydrophilic, tight microstructure for highest possible retention with lowest possible protein adsorption • For use when concentrating or desalting extremely dilute solutions or whenever your sample is hydrophobic

High Flow Biomax Ultraltration Membranes • Open microstructure speeds concentration • To concentrate and desalt higher volumes or more concentrated samples

Ordering Information Ultracel Ultrafiltration Discs Regenerated Cellulose Filter Diameter (mm)

NMWL Qty/Pk

1,000

3,000

5,000

10,000

30,000

25

10

PLAC 025 10

PLBC 025 10

PLCC 025 10

PLGC 025 10

PLTK 025 10

PLHK 025 10

44.5

10

PLAC 043 10

PLBC 043 10

PLCC 043 10

PLGC 043 10

PLTK 043 10

PLHK 043 10

47

10

PLAC 047 10

PLBC 047 10

PLCC 047 10

PLGC 047 10

PLTK 047 10

PLHK 047 10

63.5

10

PLAC 062 10

PLBC 062 10

PLCC 062 10

PLGC 062 10

PLTK 062 10

PLHK 062 10

76 90

10 5

PLAC 076 10 PLAC 090 05

PLBC 076 10 PLBC 090 05

PLCC 062 10 PLCC 090 05

PLGC 076 10 PLGC 090 05

PLTK 076 10 PLHK 090 05

PLHK 076 10 PLHK 090 05

150

5

PLAC 150 05

PLBC 150 05

PLCC 150 05

PLGC 150 05

PLTK 150 05

PLHK 150 05

Biomax Ultrafiltration Discs Polyethersulfone Filter Diameter (mm)

NMWL Qty/Pk

5,000

10,000

30,000

50,000

100,000

25 44.5

10 10

PBCC 025 10 PBGC 025 10 PBCC 043 10 PBGC 043 10

PBTK 025 10 PBTK 043 10

PBQK 025 10 PBQK 043 10

PBHK 025 10 PBHK 043 10

47

10

PBCC 047 10

PBGC 047 10

PBTK 047 10

PBQK 047 10

PBHK 047 10

63.5 76

10 10


PBTK 062 10 PBTK 076 10

PBQK 062 10 PBQK 076 10

PBHK 062 10 PBHK 076 10

90 150

5 5


PBTK 090 05 PBTK 150 05

PBQK 090 05 PBQK 150 05

PBHK 090 05 PBHK 150 05

100,000

s c s i D n o i t a r t l i f a r t l U • N O I T A R T L I F A R T L U R O F S E C I V E D D N A S E N A R B M E M

31

S E C I V E D D N A S E N A R B M E M G N I T C E L E S O T E D I U G

32

Protocols for Proteins

Concentration, Desalting and Buffer Exchange. 34 Detergent Removal . . . . . . . . . . . . . . . . . . . . 38 Two-Dimensional Electrophoresis Sample Preparation . . . . . . . . . . . . . . . . . . . . 40 Purification of Serum Peptides for Biomarker . . 42 Rapid Antibody Concentration . . . . . . . . . . . . 45 Affinity Purification . . . . . . . . . . . . . . . . . . . . 46 Removal of Unincorporated Label from Labeled Protein . . . . . . . . . . . . . . . . . . . 50 Rapid Purification of Monoclonal Antibodies . . 52 A Simple Strategy for Protein Fractionation . . . 54 Urine Concentration. . . . . . . . . . . . . . . . . . . . 56 Use of Centrifugal Filter Devices as an Alternative to Stirred Cells . . . . . . . . . . . . . . . 58

S N I E T O R P R O F S L O C O T O R P

33

Concentration, Desalting, and Buffer Exchange with Amicon Ultra or Microcon Centrifugal Filters

e g n a h c x E r e f f u B d n a g n i t l a s e D , n o i t a r t n e c n o C • S N I E T O R P R O F S L O C O T O R P

34

Introduction

Method

Amicon centrifugal devices from Millipore are ideal for removal or exchange of salts, sugars, nucleotides, and non-aqueous solvents, as well as other materials of low molecular weight. They also serve to separate free from bound species. Millipore centrifugal concentrators provide fast, convenient, high-recovery alternatives to dialysis and ethanol precipitation. Sample dilution, often associated with spin columns, is not a problem. Salt transfer across the membrane is efficient and independent of microsolute concentration or size. Millipore’s Amicon Ultra-4 and -15 centrifugal filters are designed for high speed with high recovery. The devices incorporate low-binding Ultracel regenerated cellulose ultrafiltration membrane for sample concentration and purification of solutions containing dilute or purified protein solutes, antigens, antibodies, enzymes, or microorganisms. Their speed and excellent recovery make them ideal for desalting and buffer exchange applications. One of the most common applications for Amicon Ultra devices is concentration and desalting of column fractions during protein purification by various chromatography methods. Two examples below demonstrate the use of Amicon Ultra devices for high protein and enzymatic activity recovery.

1. Select the device with the appropriate NMWL and volume for the application. 2. Add the sample to the reservoir of the centrifugal device. 3. If the sample is smaller than the maximum volume, it can be diluted up to the maximum volume before the first centrifugation step. This will help increase the salt removal. 4. Centrifuge at the specified g-force for the recommended amount of time. 5. Remove the initial filtrate from the filtrate tube and set aside. 6. Add enough buffer or water to the device to bring the sample volume up to 4 or 15 mL. 7. Centrifuge again. 8. Set aside the filtrate. 9. Recover the concentrated, de-salted sample. NOTE: Both of the filtrates should be retained until the concentrated sample has been analyzed.

Results The transfer of salts across a membrane filter is independent of sample concentration or size. There is no change in the composition of the buffer when desalting using ultrafiltration. For example, a solution containing 500 mM salt still contains that concentration after the initial centrifugation. Adding another volume of salt-free buf fer or water to the retentate and centrifuging again will reduce the salt concentration. This process, known as diafiltration, can be repeated to achieve maximum salt removal. Diafiltration can also be used when it is desirable to have the sample in a different buffer. The sample is concentrated and then repeatedly diluted with the desired buffer and concentrated again.

As the results show in Tables 1 and 2, the efficient design of the Millipore devices allowed >90% of the salt to be removed during the first centrifugation step. Typically, only one subsequent centrifugation step was needed to increase the typical salt removal to 99% with >90% recovery of the sample. Protein purification by chromatography usually involves the collection of multiple column fractions, with only some of those fractions containing the protein of interest. After the fractions are combined, a protein concentration step is of ten required for protein storage, or concentration with buffer ex-change may be needed for downstream separations.

Concentration of Indoleamine 2,3-Dioxygenase Courtesy of Eduardo Vottero, University of British Columbia Indoleamine 2,3-dioxygenase (IDO; MW 48,000) is a heme-containing enzyme that is the first and rate-limiting enzyme in human tryptophan metabolism. IDO processes 98% of the total tryptophan available in the human body and is critical in suppression of immunoresponse by blocking T-lymphocyte proliferation locally [Swanson et al, Am J Respir Cell Mol Biol [manuscript in preparation] (2003); Sarkhosh et al, J Cell Biochem 90, 206 (2003); Mellor et al, J Immunol 171, (2003)].

Table 1. Removal of sodium chloride and recovery of protein with Amicon Ultra-15 and Ultra-4 devices

NMWL

Spin 1 2

Cytochrome c 0.25 mg/mL

Cytochrome c 0.25 mg/mL

BSA 1 mg /mL

BSA 1 mg/mL

IgG 1 mg/mL

5 kDa

10 kDa

30 kDa

50 kDa

100 kDa

% Protein % NaCl Recovery Removal 94.5 97.2 92.6 99.9





Three Amicon Ultra-15 devices of each cut-off were tested with 15 mL of solute. 500 mM NaCl was added to each solution. Each spin was performed at 4000 x g for 30 minutes. After the first spin, the retentate was brought up to 15 mL with ultrapure water from a Milli-Q ® (Millipore) system. OD readings were taken at 410 nm for Cytochrome c and 280 nm for BSA and IgG.

Table 2. Removal of riboflavin and recovery of IgG with Microcon filter device with Ultracel membrane Spin Number

% NaCI Remaining

% IgG Recovered

1 2 3

9 2 <1

95 93 93

4

<1

92

500 µL of a 50:50 mixture of riboflavin and IgG were spun in a Microcon 3K NMWL device at 12,000 x g for 75 minutes at room temperature in 55° angle rotor. After the initial spin, the retentate was twice diluted with 500 µL of PBS and spun again. After each spin, concentration of riboflavin and IgG in the filtrate and retentate were monitored.


35

Recombinant IDO was expressed in E. coli BL21 (DE3) cells utilizing the pET 28a (+) vector system. In this system, a hexahistidyl tag was fused to fullsize IDO at the N-terminus with a spacer sequence and a thrombin cleavage site. The protein was purified by conventional His-tag purification methods and eluted with imidazole. The histidine tag was removed by thrombin cleavage. Final purification was done by gel filtration chromatography G-75. Amicon Ultra-15 centrifugal devices were used to concentrate the IDO fractions from an initial concentration of ~0.5 mg/mL to a final concentration of 10 mg/mL. Samples were analyzed by SDS-PAGE using a 12.5% polyacrylamide gel (Figure 1). In addition, it was shown that no IDO activity loss was observed after concentration using an Amicon Ultra device.

developed for PKR, and PKR has been purified using three chromatography steps on Agarose-Heparin, Agarose-Poly (I), Poly (C) and Sephacr yl® S-200 gel filtration columns. After the last step, PKR-containing column fractions were pooled and concentrated using Amicon Ultra-15 30K NMWL devices. The concentration step was necessary for long-term protein storage. Table 3 shows the protein recovery results obtained after four concentrations. Over 90% recovery was obtained and no protein loss to the filtrate was observed. Amicon Ultra-15 30K NMWL devices were also used for exchanging buffer for PKR autophosphorylation (activation) assay. 200 µL of 6.301 mg/mL PKR in Protein Storage buffer (20 mM HEPES, 1 M NaCl, 10 mM b-mercaptoethanol, 0.1 mM EDTA, 10% glycerol, pH 7.5) was diluted to 15 mL with Phosphorylation Buffer (20 mM HEPES, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, pH 7.5) and re-concentrated three times using

Concentration of PKR and Buffer Exchange e g n a h c x E r e f f u B d n a g n i t l a s e D , n o i t a r t n e c n o C • S N I E T O R P R O F S L O C O T O R P

36

Courtesy of Peter A. Lemaire and Dr. James Cole, University of Connecticut Human protein kinase R (PKR) is one of the major proteins induced by interferon as part of the host defense against viral infection1–4. PKR is synthesized in a latent form and is activated by autophosphorylation induced upon binding dsRNA. Once phosphorylated, active PKR phosphorylates the eukaryotic translation initiation factor elF2a leading to a block in protein synthesis in virally infected cells. PKR has been implicated as a participant in various signal transduction pathways associated with cellular processes including transcription7–9, differentiation10, apoptosis11, splicing14 and transformation5,6. However, difficulties in purifying PKR in large amounts has limited rigorous biophysical characterization of the mechanisms of PKR activation. A high yield prokaryotic expression system has been

Figure 1.

40 kDa 35 kDa

SDS-PAGE of purified indoleamine 2,3-dioxygenase before and after concentration using Amicon Ultra-15 centrifugal devices.

Table 3. PKR concentration results Spin 1

Spin 2

Spin 3

Spin 4

Starting Volume (mL) Starting Concentration (mg/mL) Volume of Retentate (µL)

15 0.229 590

15 0.229 490

15 0.169 203

15 0.169 225

Total Amount of PKR (mg) Before UF After UF Filtrate % Recovery

3.4350 3.1005 0.0000 90.26

3.4350 3.1548 0.0000 91.84

2.5320 2.4081 0.0000 95.11

2.5320 2.3983 0.0000 94.72

Amicon Ultra devices at 3000 x g for 20 minutes at 4 °C. The filtrates from the three steps were pooled and the total amount of protein in all samples was determined by UV absorption A280. Protein activity was tested by autophosphorylation assay. The protein in the storage buffer was supplemented with 5 mM MgCl2 and the samples were allowed to undergo autophosphorylation at 30 °C for 20 minutes in the presence of 3 mM ATP and 3 µCi [g 32P] ATP. The activity was determined by autoradiography and quantified by liquid scintillation counting. As shown in Figure 2, the activity of PKR when no buffer exchange was only 6% of that when buffer exchange step was performed. Hence, the UF successfully exchanged the buffer while maintaining the activity of the protein.

Figure 2. 100 R 80 K P o y t i 60 v i t c A e 40 v i t a e R 20

0

No Buffer Exchange

Buffer Exchange

Comparison of PKR autophosphorylation with buffer exchange by ultrafiltration and without buffer exchange.

References 1. Samuel CE. Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology 1991;183(1):1–11.

8. Cuddihy AR, et al. Double-stranded-RNAactivated protein kinase PKR enhances transcriptional activation by tumor suppressor p53. Mol Cell Biol 1999;19(4):2475–84.

2. Hovanessian AG. The double stranded RNAactivated protein kinase induced by interferon: dsRNA-PK. J Interferon Res 1989;9(6):641– 7.

9. Demarchi F, Gutierrez MI, Giacca M. Human immunodeficiency virus type 1 Tat protein activates transcription factor NF-kappaB through the cellular interferon-inducible, doublestranded RNA-dependent protein kinase, PKR. J Virol 1999; 73(8):7080–6.

3. Lebleu B, et al. Interferon, double-stranded RNA, and protein phosphorylation. Proc Natl Acad Sci USA 1976;73(9):3107–11. 4. Samuel CE. Mechanism of interferon action: phosphorylation of protein synthesis initiation factor eIF-2 in interferon-treated human cells by a ribosome-associated kinase processing site specificity similar to hemin-regulated rabbit reticulocyte kinase. Proc Natl Acad Sci USA 1979;76(2):600–4. 5. Koromilas AE, et al. Malignant transformation by a mutant of the IFN-inducible dsRNAdependent protein kinase. Science 1992; 257:1685–9. 6. Meurs E, et al. Tumor supressor function of interferon-induced double-stranded RNA activated protein kinase. Proc Natl Acad Sci USA 1993;90:232–6. 7. Wong AH, et al. Physical association between STAT1 and the interferon-inducible protein kinase PKR and implications for interferon and double-stranded RNA signaling pathways. EMBO J, 1997;16(6):1291–304.

10. Petryshyn R, et al. Effect of interferon on protein translation during growth stages of 3T3 cells. Arch Biochem Biophys 1996;326(2):290–7. 11. Barber GN. Host defense, viruses and apoptosis. Cell Death Differ 2001;8(2):113–26. 12. Tan SL, Katze MG. The emerging role of the interferon-induced PKR protein kinase as a apoptotic effector: A new face of death? J Interferon Cytokine Res 1999;19:543–54. 13. Balachandran S, et al. Activation of the dsRNA-dependent protein kinase, PKR, induces apoptosis through FADD-mediated death signaling. EMBO J, 1998;17(23):6888–902. 14. Osman F, et al. A cis-acting element in the 3’untranslated region of human TNF-alpha mRNA renders splicing dependent on the activation of protein kinase PKR. Genes Dev 1999;13(24): 3280–93.


37

Detergent Removal with Microcon and Amicon Ultra Centrifugal Filters

l a v o m e R t n e g r e t e D • S N I E T O R P R O F S L O C O T O R P

38

Introduction

Method and Results

Microcon and Amicon Ultra centrifugal filters are efficient laboratory tools for removing small molecules from solutions of proteins or nucleic acids. Often, the molecule to be removed is one of a number of commonly used detergents or protein solubilizing agents. The chemical nature of most detergents allows for micelle formation above a critical concentration limit (Critical Micelle Concentration, CMC). Micelle formation results in aggregation of the detergent and leads to gross changes in molecular structure. This affects the amount of the detergent that can be removed from a solution by centrifugal devices with specific nominal molecular weight limit (NMWL) membranes. For example, the monomer of Triton® X-100 has a molecular weight of 500–650 daltons. Triton X-100 should pass readily through the 10,000 NMWL membrane in an Amicon Ultra device. However, at concentrations above 0.01% (0.2 mM), Triton X-100 forms micelles comp osed of approximately 140 monomeric units. During ultrafiltration, the micelles behave like 70,000– 90,000 dalton globular proteins. As a result, more than 90% of Triton is retained by the ultrafiltration membrane. Therefore, above the CMC of Triton X-100, an Amicon Ultra-4 100K NMWL concentrator would be required to remove the detergent effectively.

In a series of studies, Millipore researchers used Total Organic Carbon (TOC) analysis to measure detergent removal using either Microcon or Amicon Ultra concentrators after a single centrifugation spin (note that complete detergent removal generally requires 3–5 spins). As the results in the Tables 1 and 2 indicate, detergent removal depends both on the original detergent concentration and the NMWL of the centrifugal units. All measurements shown were made using detergent/ distilled water solutions. NOTE: Temperature, the presence of salts in the solution, and/or macromolecule/detergent interactions may lower the CMC for a particular detergent. Use the tables only as general guidelines in assessing the efficiency of detergent removal with the Microcon and Amicon Ultra devices.

Table 1. Percent detergent removal after one spin with Microcon centrifugal devices

Table 2. Percent detergent removal after one spin with Amicon Ultra-4 centrifugal devices

NMWL Detergent (%)

SDS 0.001 0.1 1 5 NaDeoxycholate 0.1 1 5 CAPS 5 CPCl1 0.01 0.1 1 5 TDMABr2 0.1 1 5 Digitonin 0.01 0.1 1 Tween®-20 0.01 0.1 1 5 Triton X-100 0.01 0.1 1 5 CHAPS 0.1 1 5 1

3 kDa

10 kDa

50 kDa

NMWL

100 kDa Detergent (%)

>90% >90% 40–89% <40%

>90% >90% 40–89% <40%

>90% >90% 40–89% <40%

>90% >90% 40–89% <40%

>90% >90% 40–89% <40%

>90% >90% 40–89%

>90% >90% 40–89%

>90% >90% 40–89%

>90% >90% >90%

>90% >90% >90%

>90%

>90%

>90%

>90%

>90%

>90% 40–89% <40% <40%

>90% 40–89% <40% <40%

>90% 40–89% <40% <40%

>90% 40–89% <40% <40%

>90% 40–89% <40% <40%

>90% <40% <40%

>90% <40% <40%

>90% <40% <40%

>90% 40–89% <40%

>90% <90% <90%

>90% 40–89% <40%

>90% 40–89% <40%

>90% 40–89% <40%

>90% 40–89% <40%

>90% 40–89% <40%

<40% <40% <40% <40%

<40% <40% <40% <40%

40–89% <40% <40% <40%

40–89% 40–89% <40% <40%

>90% >90% 40–89% <40%

40–89% <40% <40% <40%

40–89% <40% <40% <40%

40–89% <40% <40% <40%

40–89% <40% <40% <40%

>90% >90% 40–89% <40%

>90% 40–89% <40%

>90% 40–89% <40%

Cetylpyridinium chloride Tetradecyltrimethylammonium bromide

2

30 kDa

>90% >90% 40–89%

>90% >90% >90%

>90% >90% >90%

SDS 0.1 1 5 Tween-20 0.1 1 5 Triton X-100 0.10 1 5 CHAPS 0.1 1 5

10 kDa

30 kDa

74% 13% 0.5%

75% 11% 2.0%

47% 36% 39%

69% 42% 47%

40% 26% 37%

70% 37% 39%

96% 62% 36%

81% 96% 80%

l a v o m e R t n e g r e t e D • S N I E T O R P R O F S L O C O T O R P

39

Two-Dimensional Electrophoresis Sample Preparation with Amicon Ultra Centrifugal Filters

n o i t a r a p e r P e l p m a S s i s e r o h p o r t c e l E l a n o i s n e m i D o w T

Two-dimensional electrophoresis (2DE) is one of the most commonly used methods in proteome analysis. Briefly, proteins are separated by their isoelectric point (first dimension separation) followed by SDSPAGE separation by molecular weight (second dimension separation). Although it is a powerful method for simultaneously displaying hundreds of proteins, 2DE presents a challenge for sample preparation. Salts and ionic detergents are common chemicals contaminating biological samples that are not compatible with 2DE separation; however they are often required to solubilize proteins from cells and tissues. The high concentration of salts combined with the relatively low protein content make samples completely unsuitable for isoelectric focusing.

Usually protein concentration is achieved by protein precipitation with acetone or TCA. The disadvantage of protein precipitation is that some of the proteins become insoluble and can not be resolubilized in IPG buffer. Another disadvantage is that many salts become insoluble in acetone and precipitate along with the proteins. Ultrafiltration (UF) can achieve protein concentration and desalting in one step. Figures 1 and 2 present two examples of protein preparation for 2DE by acetone precipitation and ultrafiltration. Both examples demonstrate that UF provides more efficient salt removal and allows better separations and improved resolution of protein spots in two-dimensional electrophoresis.

Figure 1.

• S N I E T O R P R O F S L O C O T O R P

40

Two-dimensional electrophoresis of yeast cell lysate prepared by acetone precipitation (left) and ultrafiltration (righ t). Sacharomyces cerevisiae strain s288c was grown to log phase. The cells were pelleted and resuspended in Cellular and Organelle Membrane Solubilizing Reagent from the ProteoPrep ™ kit (Sigma) and lysed by sonication. Cellular debris was removed by centrifugation and the supernatants were reduced and alkylated with 5 mM tributylphosphine and 10 mM acrylamide. Lysates were acetone-precipitated to remove residual Tris and alkylating reagent or were filtered through Amicon Ultra-4 10K NMWL devices. Samples were redissolved in ProteomIQ ™ Resuspension Reagent (Proteome Systems), focused in broad range (pH 3–10) immobilized pH gradients (IPGs) on the IsoelectrIQ ™ IEF instrument. Second dimension gels were 6–15% polyacrylamide gradient GelChips™ (Proteome Systems) run on an ElectrophoretIQ ™ 2D instrument (Proteome Systems). Data courtesy of Dr. G. Smejkal, Proteome Systems, Inc., Woburn, MA, USA.

Figure 2.

Two-dimensional electrophoresis of endothelial cell lysates prepared by acetone precipitation (left) and ultrafiltration with Amicon Ultra devices (right). Whole cell lysates of endothelial cells were prepared in 7 M urea, 2 M thiourea, 4% CHAPS, 10 mM DTT, 20 mM Tris buffer. The samples were very dilute and presumably contained DNA fragments, salts and lipids. 300 mg of the total protein was either acetone-precipitated or desalted in Amicon Ultra devices. After acetone precipitation with four volumes of cold acetone, samples were incubated overnight at –20 °C, precipitated by centrifugation, rinsed with cold acetone, and pelleted again. The resulting pellet was resuspended in rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 10 mM DTT, 20 mM Tris). Alternatively, samples were filtered through Amicon Ultra-4 10K NMWL devices after being mixed with nine volumes of 20 mM Tris HCl, pH 7. The sample was concentrated to approximately 40 µL and adjusted to 350 µL with rehydration buffer. The proteins were separated by IEF in 18 cm IPG Drystrips ™ , pH 3–10 (GE Healthcare). A second dimension separation was performed in 10% SDS-PAGE gel. The gels were silver stained and scanned. Data courtesy of Dr. Leonid Kry azhev, Genome Quebec, Montreal, Canada.

n o i t a r a p e r P e l p m a S s i s e r o h p o r t c e l E l a n o i s n e m i D o w T • S N I E T O R P R O F S L O C O T O R P

41

Purification of Serum Peptides for Biomarker Research with Amicon Ultra Centrifugal Filters

h c r a e s e R r e k r a m o i B r o f s e d i t p e P m u r e S f o n o i t a c i f i r u P • S N I E T O R P R O F S L O C O T O R P

42

Introduction

Methods

A biomarker can be defined as a molecule that indicates an alteration in physiology. Biomarkers play an essential role in the drug discovery and development process. They provide powerful clues to genetic susceptibility, disease progression, and predisposition, as well as drug response and the physiological and metabolic profiles of diseases and drug responses. Biomarkers can also provide valuable diagnostic and prognostic information that can facilitate personalized medicine. Peptide and protein patterns have been linked to ovarian cancer, breast cancer, prostate cancer and astrocytoma1–5. Most diagnostic tests are based on blood or urine analysis5. Serum is a key source of putative protein biomarkers, and, by its nature, can elucidate organ-confined events. Mass spectrometry, coupled with bioinformatics, is capable of distinguishing between serum protein pattern signatures in latestage and early-stage ovarian cancer patients6. One of the major impediments to the discovery of new biomarkers is the presence of salts, prot eins, and lipids in plasma or serum that makes it difficult to detect and analyze peptides by mass spectrometry. When untreated serum is spotted onto a MALDI-TOF plate, it does not produce any usable signal in mass spectrometry. Multiple protocols have been developed to extract and enrich peptides from tissues and body fluids, such as batch reversed phase chromatography over C18 resin and extraction with 0.1% trifluoroacetic acid (TFA) or 50% acetonitrile to selectively precipitate large prot eins while enhancing the solubility of smaller proteins and peptides. Ultrafiltration has previously been reported as a sample preparation tool to prepare low molecular weight fractions for biomarker analysis7–10. In this study we show that ultrafiltration in combination with solid phase extraction (SPE) on C18 resin can be a convenient and efficient method for serum peptide purification. The approach provides more peptides for mass spectrometry analysis than the acetonitrile precipitation method.

Preparation of Serum Peptides by UF and SPE One milliliter of human serum, with or without acetonitrile, was filtered using Amicon Ultra-4 10K NMWL centrifugal devices. The devices were centrifuged in a swinging bucket rotor for 15 to 30 minutes at 3000 x g. Ten microliters of the filtrate was acidified with 5 µL of 1% TFA, concentrated with ZipTip®µC18 pipette tips. Co-elution was performed directly onto a MALDI target with 2 µL of ∂-cyano-4-hydroxycinnamic acid matrix (5 mg/mL in 50% acetonitrile, 0.1% TFA). If acetonitrile was added to the serum prior to the filtration, the samples were briefly evaporated in a Speed Vac® centrifuge to remove solvent before ZipTip purification.

Peptide Analysis by Mass Spectrometry Peptide-containing ultrafiltrates from cell lysates or human serum were acidified with 1% TFA and concentrated on ZipTipµC18 or ZipTipSCX pipette tips following the procedure outlined in the user guide. All samples were overlaid with 1 µL of ∂-cyano4-hydroxycinnamic acid matrix (5 mg/mL in 50% acetonitrile, 0.1% TFA) and analyzed on Voyager-DE ™ Workstation (Applied Biosystems) in linear mode.

Preparation of Serum Peptides by Acetonitrile Precipitation Acetonitrile was added to human serum at a 1:1 ratio (v:v), and samples were centrifuged to precipitate larger proteins. The supernatant was dried in a Speed-Vac centrifuge, resuspended in 0.1% TFA, and then desalted and concentrated with ZipTipµC18 pipette tips. Co-elution was performed directly onto a MALDI target with 2 µL of ∂-cyano4-hydroxycinnamic acid matrix (5 mg/mL in 50% acetonitrile, 0.1% TFA).

Results While human serum contains numerous peptides and small proteins, they are not accessible by direct mass spectrometry analysis. Even after reverse phase concentration and desalting, only a few peptides are detectable in the mass spectrum (data not shown). This can be explained by the high concentration of proteins and lipids competing with the peptides to bind to the resin (Figure 1A).

One of the common methods for serum peptide preparation for mass spectrometry is acetonitrile fractionation, where the addition of 50–70% acetonitrile precipitates larger proteins, while the peptides stay soluble in the supernatant. Another way to produce relatively protein-free filtrates is ultrafiltration. Figure 2 presents the MALDI-TOF spectra of (A) straight rat serum; (B) serum supernatant after 50% acetonitrile precipitation; and (C)

Figure 1. A

B

C

MALDI-TOF spectra of (A) unprocessed rat serum; (B) serum peptides in 50% acetonitrile supernatant; and (C) serum ultrafiltrate processed with Amicon Ultra-4 30K NMWL centrifugal device.

Figure 2. A

B

C

D

h c r a e s e R r e k r a m o i B r o f s e d i t p e P m u r e S f o n o i t a c i f i r u P •

MALDI-TOF spectra of rat serum peptides after concentration and desalting by reversed phase chromatography: (A) rat serum processed with ZipTip µC18 pipette tip; (B) serum supernatant after 50% acetonitrile pr ecipitation processed with ZipTip µC18 pipette tip; (C) 30K serum ultrafiltrate processed with ZipTip µC18 pipette tip; and (D) the same as (C) but 20% acetonitrile was added to the serum prior to ultrafiltration.

S N I E T O R P R O F S L O C O T O R P

43

h c r a e s e R r e k r a m o i B r o f s e d i t p e P m u r e S f o n o i t a c i f i r u P • S N I E T O R P R O F S L O C O T O R P

44

serum ultrafiltrate processed with an Amicon Ultra-4 30K NMWL device. We observed higher quality spectra and an increased number of MALDI-TOF detected peptides if the serum was ultrafiltered. Further improvement of spectra can be achieved by reverse phase chromatography, which concentrates and desalts the peptides. The ZipTipµC18 pipette tip is a convenient and efficient tool for micro-scale sample preparation prior to mass spectrometry. Figure 2 shows the MALDI-TOF spectra of rat serum peptides prepared with ZipTipµC18 pipette tip out of (A) straight serum, (B) 50% acetonitrile supernatant and (C) serum processed by ultrafiltration. The last method provided stronger MALDI-TOF signal, higher signal-to-noise ratio and double the number of detected peptides. The addition of 5 to 10 % acetonitrile to serum and plasma samples (data not shown) prior to ultrafiltration was shown to improve the detection of serum peptides, the MALDI spectrum and the overall signal intensity.

Conclusion For the analysis of serum peptides, complexity reduction by eliminating higher molecular weight proteins is critical for high resolution mass spectrometry. Efficient separation of peptides from the majority of proteins and salts can be achieved by sample ultrafiltration. We have shown the effective use of Amicon Ultra-4 30K NMWL centrifugal devices for the preparation of peptides. Other molecular weight cut-offs can be utilized depending on the desired range of peptides. The method can be used directly in combination with ZipTipµC18 pipette tips for peptide identification by MS/MS or as a first step prior to further surface-mediated enrichment using SELDI-TOF methods. These sample preparation protocols may also be applicable to other low molecular weight markers such as drugs and metabolites. Peptide purification using ultrafiltration to de-proteinize followed by SPE to de-salt serum and plasma samples (data not shown) was easily adapted to the 96-well format allowing large numbers of samples to be simultaneously prepared.

References 1. Ardekani AM, Liotta LA, Petricoin III. Expert Rev Mol Diagn 2002;2:12. 2. Carter D, Douglass JF, Cornellison CD, Retter MW, Johnson JC, Bennington AA, Fleming TP, Reed SG, Houghton RL, Diamond TS, Vedvick TS. Biochemistry 2002;41:6714. 3. Wellmann A, Wollscheid V, Lu H, Ma ZL, Albers P, Schutze K, Rohde V, Behrens P, Dreschers S, Ko Y, Wernert N. Int J Mol Med 2002;9:341. 4. Petricoin EF, Ardekani AM, Hitt BA, Levine PJ, Fusaro VA, Steiberg SM, Mills GB, Simone C, Fishman DA, Kohn EC, Liotta LA. Lancet 2002; 359:572. 5. Bischoff R, Luider TM. J Chrom B 2004;803: 27–40. 6. Stevens EV, Liotta LA, Kohn EC. J Gynecol Cancer 2003;13:133–9. 7. Schulz-Knappe P, Schrader M, Standker L, Richter R, Hess R, Jurgens M, Forssmann W-G. J Chromatogr A 1997;776:125–132. 8. Basso D, Valerio A, Seraglia R, Mazzza S, Piva MG, Greco E, Fogar P, Gall N, Pedrazzoli S, Tiengo A, and Plebani M. Pancreas 2002;24:8–14. 9. Prazeres S, Santos MA, Ferreira HG, Sobrinho LG. Clin Endocrinol (Oxf) 2003;58:686–90. 10. Tirumalai RS, Chan KC, Prieto DA, Issaq HJ, Conrads TP, Veenstra TD, Mol Cell Proteomics 2003;10:1096–103. 11. Chernokalskaya E, Gutierrez S, Pitt AM, Leonard J. Electrophoresis 2004;25: 2461–2468. 12. Millipore Case Study PR0001EN00. Automating Multiplexed Cytokine Assays manuscript in preparation.

Rapid Antibody Concentration with Amicon Ultra Centrifugal Filters Ultrafiltration offers a fast and convenient way to concentrate antibodies purified from serum, ascites fluid, or hybridoma supernatants. The traditional purification protocol for immunoglobulins includes affinity binding to Protein A or G chromatography media, washing unbound proteins, and eluting with a low pH buffer. Subsequently, purified antibodies are often too dilute for their intended purpose or for long-term storage. In addition, harsh elution conditions sometimes require buffer exchange to preserve protein activity. Dialysis is often used for antibody concentration and buffer exchange. However, ultrafiltration provides a quick, alternative method to concentrate and diafilter immunoglobulins with up to 99% recovery and one-step salt removal (see Concentration, Desalting, and Buffer Exchange with Amicon Ultra or Microcon Centrifugal Filters, page 34). To demonstrate the suitability of Amicon Ultra devices for concentrating purified IgG, rabbit serum was processed with Montage Purification Kits with PROSEP®-A and PROSEP-G Antibody (Millipore) and then concentrated with Amicon Ultra-15 30K NMWL devices. Figure 1 shows a decrease in the retentate volume proportional to the increase in antibody concentration. A twenty-minute centrifugation resulted in >95% recovery of immunoglobulins. Figure 2 shows an SDS-PAGE gel of purified rabbit IgGs before and after ultrafiltration.

Figure 1. 40

12 PROSEP-A

10

PROSEP-G

30 ) L m ( e m u 20 l o V G g I

8 6 4

10 2 0

) L m / g m ( n o i t a r t n e c n o C G g I

0 0

3

10

15

20

Centrifugation Time (min) Concentration of rabbit IgG with Amicon Ultra-15 devices. The IgGs were purified using Montage PROSEP-A or PROSE P-G Antibody Purification Kits. The lines show IgG volume reduction, while the bars show a proportional increase in IgG concentration after 20 minutes of centrifugation time.

Figure 2. SDS-PAGE gel of purified rabbit IgG before and after concentration with Amicon Ultra-15 devices.

Lane 1: Lanes 2, 3:

MW standards PROSEP-A-purified IgG before concentration (lane 2) and after concentration (lane 3) Lanes 4, 5: PROSEP-G-purified IgG before concentration (lane 4) and after concentration (lane 5) Protein Load: 5 µL in lanes 2 and 4; 1 µL in lanes 3 and 5

kDa 116

66 55

1

2

3

4

5

Heavy Chain

36

21 14

Light Chain

n o i t a r t n e c n o C y d o b i t n A d i p a R • S N I E T O R P R O F S L O C O T O R P

45

Affinity Purification with Ultrafree-MC Centrifugal Filters Introduction Affinity interaction chromatography is often the single most effective step in any protein purification procedure. Up to 95% purity can be achieved in one step, depending on the nature of the interaction and the starting composition of the protein solution. Well known examples of highly specific affinity interactions include antibodies and protein A/G; multiple histidine tags and nickel; streptavidin and biotin; antibodies and antigens; and many others. Less specific interactions are also used for enrichment or depletion protocols, including albumin depletion on cibacron blue resin, glycoprotein enrichment on concavalin A resin, and capture of nucleic acid-binding proteins on heparin resin. Affinity chromatography is often employed in the small-scale batch mode as a quick method for microgram-scale protein purification. The typical protocol involves: 1. Pipetting a small volume of affinity resin into a microfuge tube that contains the sample n o i t a c i f i r u P y t i n i f f A • S N I E T O R P R O F S L O C O T O R P

46

2. Vortexing the tube for a few minutes 3. Centrifuging the resin to the bottom 4. Pipetting off the supernatant 5. Washing a few times (using steps 2 and 3) 6. Eluting with a small amount of eluant Although the method is relatively simple, care must be taken not to remove the chromatography resin when pipetting off the supernatant. Pre-packed mini-spin columns are a convenient tool for small-scale protein purification. Operated by centrifugation, they usually complete the whole procedure in less than an hour.

Ultrafree-MC centrifugal filter unit

Another alternative for small-scale purification are centrifugal devices with microporous membrane, such as Ultrafree-MC centrifugal devices. The sample can be added to the filter basket and mixed for the needed residence time and then centrifuged. The process removes the interstitial liquid but does not dehydrate the beads. Washing and elution can also be performed in a similar manner and are more effective due to the efficient removal of buffer and/or eluant. Ultrafree-MC centrifugal filter units with microporous membrane come with low protein-binding Durapore PVDF membrane in five different pore sizes from 0.1 to 5.0 µm. Affinity resin can be loaded into the filter basket and the device used as a “home-made” mini-spin column. We show the applicability of the device for purification of rabbit IgG on PROSEP-A resin and His-tagged C-RP protein on three different commercial metal-chelate resins.

Materials

Method for IgG P urification

• Ultrafree-MC 0.45 µm centrifugal devices (Millipore cat. no. UFC3 0HV 00)

Solutions

• PROSEP-A high capacity resin (Millipore cat. no. 1131 118 26) • Rabbit serum Gibco (Invitrogen lot no. 1132782) ®

• Micro-centrifuge Biofuge Pico (Heraeus instruments) • Jouan CR1822 fixed angle rotor centrifuge • Xcell SureLock ™ Mini-cell vertical electrophoresis system (Invitrogen cat. no. EI0001) ®

• NuPage NOVEX Bis-Tris 4 –12%, 1 mm thick, 15 well SDS gels, (Invitrogen cat. no. NP0323) • NuPage Sample Reducing agent (10X) (Invitrogen cat. no. NP009) • NuPage SDS Sample Buffer (4X) (Invitrogen cat. no. NP007) • SimplyBlue™ SafeStain Coomassie G-250 stain (Invitrogen cat. no. LC6060)

• PROSEP-A binding buffer A: 1.5 M Glycine/ NaOH, 3 M NaCl, pH 9.0 • PROSEP-A elution buffer B2: 0.2 M Glycine/ HCl, pH 2.5 • PROSEP-A neutralization buffer: 1 M Tris/HCl, pH 9.0

Procedure 1. 200 mg of PROSEP-A media were placed in Ultrafree-MC 0.45 µm filter basket. 2. The columns were equilibrated with 400 µL of binding buffer A and centrifuged for 1 minute at 100 x g. 3. 200 µL of rabbit serum were diluted 1:1 with binding buffer and the entire volume was loaded into the spin column containing PROSEP-A resin. 4. Devices were placed on a shaker for 15 minutes at room temperature and centrifuged at 100 x g for 5 minutes. Flow-through was collected for future analysis. 5. Three consecutive washes of 400 µL each were performed by adding 400 µL of binding buffer A and centrifuging at 2,000 x g for 2 minutes each. 6. 200 µL of elution buffer B2 were added and centrifuged for 2 minutes at 2,000 x g. 7. 26 µL of neutralization buffer were added to each collection tube. A second elution was collected after repeating the same process one more time.

n o i t a c i f i r u P y t i n i f f A • S N I E T O R P R O F S L O C O T O R P

47

Method for His-tagged C-RP Purification Solutions • Lysis buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM immidazole, pH 7 • Binding buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM immidazole, pH 7 • Wash buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 20 mM immidazole, pH 7 • Elution buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 250 mM immidazole, pH 7 • 1 mg/mL Lysozyme stock

5. 500 µL of the clarified lysate were added to the resin. 6. The His-tagged proteins were bound for 30 minutes with light agitation. 7. The lysate was removed by centrifugation at 500 x g for 10 minutes. 8. The resin was washed with 500 µL of wash buffer for 5 minutes with agitation. The wash solution was removed by centrifugation for 5 minutes at 500 x g. This step was repeated two more times. 9. 250 µL of elution buffer were added to the Ultrafree-MC device and mixed for 5 minutes. Purified protein was recovered by centrifugation at 500 x g for 1 minute.

• Benzonase

Procedure 1. Recombinant proteins were expressed in Escherichia coli . 2. Cells were prepared at a 10X concentration using lysis buffer. Lysozyme was added to a concentration of 0.1 mg/mL. To reduce the viscosity, benzonase was added to t he lysate. The lysates were clarified by centrifugation.


48

3. 200 µL of the 50% resin slurry were added to the Ultrafree-MC device and the residual fluid was removed by centrifugation for 1 minute at 500 x g. 4. The resin was equilibrated with 500 µL of binding buffer and centrifuged for 2 minutes at 500 x g.

Results The results of purifying rabbit IgG using UltrafreeMC centrifugal devices are shown in Figure 1. The device was challenged with approximately 14 mg of total protein, with an estimated IgG content of 1.5–2 mg. The original serum, flowthrough, three washes and two eluted fractions were analyzed by SDS-PAGE. The total amount of purified IgG, as estimated by OD280, was 1.2 mg and 1.1 mg on two devices processed in parallel. The whole procedure was completed in less than one hour. This method can be useful for monitoring the titer of antigen-specific antibodies after immune activation, or whenever small amounts of IgG need to be purified.

Figure 2 shows the results of His-tagged protein purification in Ultrafree-MC devices using Ni-NTA agarose and BD Talon™ resin. Two recombinant proteins were purified: C-RP, high-expressing protein of 26 kDa; and RT66, low-expressing protein of 66 kDa. Both purifications resulted in high purity proteins. The amount of proteins purified out of 500 µL of lysate was 32–35 µg for C-RP protein and 8 µg for RT66. The data show that affinity batch purification can be effectively performed on a small scale using Ultrafree-MC devices loaded with resin. The process combines the high efficiency of batch binding and washing with the handling convenience of a mini-spin column. With minimal handson time, the method provides flexibility of resin to lysate ratio and binding conditions, independent of centrifugation speed and rotor angle. This method is applicable to recombinant protein purification, antibody purification and immunoprecipitation.

Figure 1. Rabbit IgG purification on PROSEP-A resin in Ultrafree-MC devices.

Lane 1:

Molecular weight standards Rabbit serum Lane 2: Flow through Lane 3: Lanes 4–6: Three consecutive washes Lanes 7, 8: Eluted IgG from two devices

kDa 200

1

2

3

4

1

2

3

4

5

6

7

8

116.3 97.4 66.3 55.4 36.5 31.0 21.5 14.4 6.0

Figure 2. His-tagged protein purification using Ultrafree-MC devices.

Lane 1:

kDa 200 116.3 97.4 66.3 55.4

Molecular weight standards E. coli lysate 36.5 Lane 2: expressing 31.0 C-RP protein 21.5 Lane 3: E. coli lysate 14.4 expressing RT66 protein 6.0 Lanes 4, 5: Proteins purified on Ni-NTA resin Lanes 6, 7: Proteins purified on BD Talon resin

5

6

7

RT66

C-RP


49

Removal of Unincorporated Label from Labeled Protein with Amicon Ultra Centrifugal Filters

n i e t o r P d e l e b a L m o r f l e b a L d e t a r o p r o c n i n U f o l a v o m e R • S N I E T O R P R O F S L O C O T O R P

50

Introduction

Results

Centrifugal devices containing ultrafiltration membranes are ideal for the removal or exchange of salts, sugars, nucleotides, non-aqueous solvents, and other materials of low molecular weight. They also serve to separate free from bound species. For the removal of unincorporated label in protein labeling applications, Millipore centrifugal devices provide fast, convenient, high-recovery alternatives to gel filtration. Sample dilution, often associated with gel filtration, is not a problem. A few rounds of diafiltration can efficiently remove unincorporated label. Two examples below demonstrate the use of Millipore centrifugal ultrafiltration devices for removal of unreacted fluorescent and isotope labels.

Figure 1 shows the SDS-PAGE gel of FITC-labeled BSA before and after each of four diafiltration cycles. Unincorporated FITC is clearly visible in the starting material and in the first filtrate. After only one cycle of ultrafiltration, the majority of the free label is removed. Subsequent cycles of filtration result in BSA that is virtually free of unincorporated FITC. Fluorescence measurement offers a more sensitive method to monitor the ultrafiltration process. Figure 2 shows the change in fluorescence of filtrate and retentate during four cycles of FITC-BSA diafiltration. The results indicate that almost 80% of free FITC can be removed after the first ultrafiltration and three rounds result in 98% removal. This demonstrates the viability of ultrafiltration as an alternative to gel filtration for cleaning up protein labeling reactions. Using an Amicon Ultra-4 device, each ultrafiltration is accomplished in 10–15 minutes and allows high recovery of target protein. While gel filtration results in diluted protein fractions, ultrafiltration offers the additional advantage of concentrating protein while removing unincorporated label.

Method for Removal of FITC from FITC-BSA 1. Bovine serum albumin was labeled with fluorescein-isothiocyanate (FITC); the free unincorporated label was not removed. 2. Two mL of FITC-labeled BSA solution at 0.5 mg/mL were loaded into two Amicon Ultra-4 10K NMWL devices and centrifuged at 3,000 x g for 10 minutes. 3. Retentates (about 50 µL each) were re-diluted to 2 mL with water and centrifuged again. This step was repeated twice. 4. After each ultrafiltration, the retentate and filtrate were analyzed by SDS-PAGE and their fluorescence measured on a SpectraFLUOR™ plate reader (Tecan) at excitation 485 nm and emission 530 nm. 5. The SDS PAGE gel was scanned on a Storm® (GE Healthcare) fluorescence scanner.

Figure 1.

Figure 2. 60,000

SDS-PAGE of FITC-labeled BSA

Lane 1: Starting material Lane 2: Retentate after first ultrafiltration Lane 3: Retentate after 2 rounds of ultrafiltration Lane 4: Retentate after 3 rounds of ultrafiltration Lane 5: Retentate after 4 rounds of ultrafiltration Lane 6: Filtrate after first ultrafiltration Lane 7: Filtrate after 2 rounds of ultrafiltration Lane 8: Filtrate after 3 rounds of ultrafiltration

50,000 ) s t i e c n n U e y c r s a e r r t i o b u i F r A (

Retentate Filtrate

40,000 30,000 20,000 10,000 0 Starting material

FITC-BSA

Spin 1

Spin 2

Spin 3

Spin 4

Centrifugation Time (min)

Fluorescence measurements of the filtrates and r etentates after each of four cycles of FITC-labeled BSA ultrafiltration. Free label was transferred through the 10,000 NMWL membrane while labeled BSA was retained and concentrated. All retentates and filtrates were volume adjusted to 2 mL prior to the measurement.

FITC

1

2

3

4

5

6

7

8

n i e t o r P d e l e b a L m o r f l e b a L d e t a r o p r o c n i n U f o l a v o m e R • S N I E T O R P R O F S L O C O T O R P

51

Rapid, Ultrafiltration-based Method for Purification of Monoclonal Antibodies Introduction

s e i d o b i t n A l a n o l c o n o M f o n o i t a c i f i r u P d i p a R • S N I E T O R P R O F S L O C O T O R P

52

Monoclonal antibodies (MAb) continue to gain importance as therapeutic and diagnostic agents for many types of cancer. The process of screening hybridoma libraries for candidate MAbs is both time consuming and labor intensive. Once a hybridoma cell line expressing a suitable MAb is established, a bench-scale purification methodology (e.g., 50 to 500 mL) must be developed to produce sufficient MAb for further characterization. A traditional method for purifying MAbs involves clarification of the hybridoma supernatant by centrifugation, followed by an ammonium sulfate precipitation to concentrate the MAbs. The precipitate is then recovered by centrifugation, resolubilized and desalted using dialysis. After these steps, the MAb is further purified using Protein A/G affinity chromatography. The purified antibody is

desalted and exchanged into a biological buffer using dialysis. The entire process typically requires several days to complete and can be particularly onerous if multiple MAbs are to be evaluated in parallel. We describe here a new and simplified method that minimizes the processing time to less than a day to obtain pure MAb.

Method The method involves clarification of the hybridoma supernatant by microfiltration using a Stericup vacuum filter cup, followed by concentration using ultrafiltration 1 with a Centricon Plus-70 device (see Table 1 and Figure 1 for method details). The MAb is further purified on protein A/G beads. The purified MAb is desalted and buffer-exchanged using ultrafiltration.

Table 1. Comparison of traditional and ultrafiltration workflows Workflow for Obtaining Concentrated Supernatant Traditional Ammonium Sulfate Precipitation 1. Start with 200 mL 2. Weigh out ammonium sulfate 3. Add ammonium sulfate to clarified supernatant with constant stirring (20-30 min). Store at 4 °C. 4. Centrifuge to recover pellet, resuspend pellet 5. Prepare dialysis membrane and check for integrity 6. Collect resuspended precipitate in dialysis tubing/device 7. Dialyze with three changes of PBS (18-24 hrs) 8. Collect dialysate in centrifuge tube(s) and centrifuge (~30 min) 9. Load on protein-G column Total time required ~24 hrs

Ultrafiltration Centrifugal 1. Start with 200 mL 2. Add supernatant to Centricon Plus-70 Device (100K MWCO) 3. Centrifuge (20–30 min) 4. Invert spin to collect sample (2 min) 5. Load on protein-G column

Total time required ~40 min

Tangential Flow Filtration (TFF) 1. Start with 1000 mL 2. Add supernatant to Labscale TFF unit (30K MWCO) 3. Apply pressure (40 psi) with constant stirring (~2 hrs) 4. Collect sample (5 min)

Total time required ~2 hrs

Results This approach was successfully used to purify an anti c-myc antibody secreted by the hybridoma clone 9E102 (Figure 1). The MAb purified by this new method performed comparably to the commercially purified MAb in downstream applications such as western blotting and ELISA (Figure 2). The data demonstrate that the new protocol is robust and delivers MAb of a high purity and yield as compared to the traditionally purified MAb. The application of ultrafiltration to MAb purification will be of considerable value to any researcher interested in screening hybridoma libraries and accelerating the purification of MAbs.

Figure 1.

C

Tr

UF

C: Clarified supernatant Tr: Supernatant precipitated with ammonium sulfate and dialyzed using 10K MWCO membrane (Spectrapor, Rancho Dominguez, CA) UF: Supernatant concentrated by ultrafiltration on Centricon Plus 70 device

Figure 2. 2 Abalone

Commercial

Traditional

UF-based

0.7 UF-based method

0.6 m m0.5 0 5 4 0.4 @ D0.3 O

Traditional method Commercially available

0.2 0.1 0.0 0

10

100

Concentration of mAB (µg/mL)

Conclusion Our data show that the combination of microfiltration and ultrafiltration is a rapid method for MAb purification and that MAb purified on Montage centrifugal columns with Protein G media is comparable to the commercially available MAb in purity, activity and cost. The Ultrafiltration-based MAb purification method, compared to the traditional method, is faster (2–3 hours versus 2 days), easier to use, and yields higher recoveries.

References

Upper panel shows activity comparison measured in a western blot between MAb (derived from HEK 293) purified using ultrafiltration method versus traditional method. Three-fold serial dilutions of HEK 293 cell nuclear extracts were run on 4 to 12% NuPAGE gels and transferred to Immobilon® -P membrane (Millipore). Blots were probed with the indicated primary antibodies and secondary anti-mouse alkaline phosphatase conjugate (Sigma, St. Louis, MO). The antibodies were detected with Immobilon Western AP substrate (Millipore) and imaged on a Kodak ® scanner. Bottom panel shows activity comparison measured in an ELISA assay. HEK 293 cells were grown on 96-well plates. After fixation and epitope-retrieval by heating for 10 minutes in a microwave, cells were permeabilized with 1% saponin (Sigma) in PBS + 2% normal donkey serum (Jackson Immunoresearch, West Grove, PA) and treated with serial dilutions of the indicated purified MAbs. The cells were then washed and treated with goat anti-mouse HRP-conjugate antibody (Sigma). The reactions were developed using a SureBlue ™ TMB HRP substrate (KPL, Gaithersburg, MD). The readings were measured on a SpectraMax ® plate reader (Molecular Devices, Sunnyvale, CA).

s e i d o b i t n A l a n o l c o n o M f o n o i t a c i f i r u P d i p a R • S N I E T O R P R O F S L O C O T O R P

1. Saha K, Case R, Wong PK. J Immunol Methods 1992;151(1-2):307-308. 2. Evan GI, Lewis GK, Ramsay G, Bishop JM. Mol Cell Biol 1985;5(12):3610-3616.

53

A Simple Strategy for Protein Enrichment Using Ultrafiltration Introduction The human plasma proteome is a diverse universe of information and it will continue to be a vast source of biomarker discovery and investigation. Ultrafiltration (UF) has been reported1–3 as a sample preparation tool to prepare low molecular weight (LMW, <10 kDa) fractions by reducing the complexity of serum proteins prior to mass spectrometr y for biomarker analysis. Traditionally, enrichment of protein solutions on the basis of size has been accomplished using gel filtration chromatography 4,5. However, gel filtration is laborious, limited by sample size and time-consuming. Another consequence of gel filtration chromatography is the significant dilution of the protein relative to the n o i t a r t l i f a r t l U g n i s U t n e m h c i r n E n i e t o r P •

Figure 1. Serial enrichment strategy Step 1: 100 kDa MWCO 40 min at 1500 x g

100 kDa Retentate

This experiment demonstrates the use of ultrafiltration devices to enrich both LMW and high molecular weight (HMW) fractions from serum. Protein separation was accomplished by serial filtration through decreasing molecular weight cutoff (MWCO) devices from 100K to 10K MWCO. This serial enrichment strategy enabled the com partmentalization of proteins by size as well as an increase in throughput as compared to directly enriching samples using a lower MWCO device.

Figure 2. Purification of Cytochrome c from a mixture containing 10% fetal bovine serum (FBS) and the compartmentalization of protein fractions using Amicon Ultra devices

100 kDa Permeate (<100 kDa)

M

S

R100 P100 R50 P50

R30 P30

Step 2: 50 kDa MWCO 30 min at 1500 x g 50 kDa

50 kDa Retentate


30 kDa

Cytochrome c (12.5 kDa)

Step 3: 30 kDa MWCO 30 min at 1500 x g

R O F

54

Method

100 kDa

S N I E T O R P

S L O C O T O R P

original concentration. These limitations can be overcome by using centrifugal ultrafiltration devices for enrichment.

30 kDa Retentate


A complex mixture is centrifuged in a 100K device. The retentate is removed and saved for later analysis. The permeate fraction is then centrifuged in a 50K device. This process is repeated for the 30K device.

M: Marker S: 10% FBS + 1 mg/mL Cytochrome c R100: 100 kDa Retentate P100: 100 kDa Permeate

Purified Cytochrome c

R50: 50 kDa Rententate P50: 50 kDa Permeate R30: 30 kDa Retentate P30: 30 kDa Permeate

A serum-protein mixture spiked with purified Cytochrome c (12.5 kDa) was centrifuged in an Amicon Ultra 4 mL device using serially decreasing MWCOs as described in Figure 1. The red, blue and green lines indicate approximate molecular weight ranges and represent the theoretical MWCOs. Colored dashed boxes represent size specific protein “compartments.”

The traditional methodology employing use of a single MWCO to remove HMW and enrich for the LMW resulted in impure fractions compared to those from serial enrichment.

References

Conclusions

3. Sobrinho LG, et al. Clin Endocrinol (Oxf) 2003;58:686-690.

1. Forssmann WG, et al. J Chromatogr A 1997;776:125-132. 2. Plebani M, et al. Pancreas 2002;24:8-14.

Protein fractionation using centrifugal ultrafiltration devices is a simple and rapid means of reducing sample complexity and compartmentalize proteins based on molecular weight. Serial enrichment is faster than a direct single device enrichment and protein fractions resulting from serial enrichment have greater purity than those from a single device method. Regenerated cellulose membranes outperform PES membranes in an enrichment paradigm. Ultrafiltration is a potential alternative to gel filtration chromatography for size based protein enrichment.

4. Werner MJ. Chromatogr 1966;25(1):63-70. 5. Kent UM. Methods Mol Bio 1999; 115:11-18.

Figure 3. Serial enrichment is more efficient than direct enrichment in terms of purity and time Serial 100 min (150–300 µL) M

Time Required:

S R100 R50 R30 R10

Figure 4. Regenerated cellulose membranes offer optimal separation compared to PES

Direct 130 min (500–600 µL) M

S

R30

R10

100 kDa

50 kDa 30 kDa

30 kDa

Light Chains 10 kDa

10 kDa

M: Marker S: 50% Adult Serum R100: 100 kDa Retentate

R50: 50 kDa Rententate R30: 30 kDa Retentate R10: 10 kDa Permeate

Direct enrichment is not recommended for unknown or highly concentrated samples. The figure shows 50% adult bovine serum enriched directly (right panel) using an Amicon Ultra-4 30K MWCO device. The device was centrifuged for more than 2 hours at 1500 x g to achieve a final volume of ~500 µL, an 8X concentration. Presence of BSA and other higher molecular weight contaminating proteins clearly show the benefits of using a serial enrichment strategy (left panel) as described in Figure 1. Permeates collected from the 30K (R10) devices were concentrated with 10K devices prior to protein visualization. An unknown protein ~12 kDa was purified using a serial method, but not direct enrichment.

100

) 90 % ( C 80 e m 70 o r 60 h c o 50 t y C 40 f o 30 y r 20 e v o 10 c e R 0

IgG (0.1 mg/mL) IgG (1.0 mg/mL)

Ultracel

PES Device A

PES Device B

Regenerated cellulose membranes (Ultracel membrane) outperform polyethersulfone (PES) membranes used for enrichment. A binary mixture containing Cytochrome c (1 mg/mL) and 155 kDa IgG (0.1 or 1.0 mg/mL) was centrifuged using 100K MWCO devices. Cytochrome c recovery was measured in the permeates. Significantly greater Cytochrome c recovery was observed using Ultracel membrane when the system was challenged with high levels of IgG (1.0 mg/mL).

n o i t a r t l i f a r t l U g n i s U t n e m h c i r n E n i e t o r P • S N I E T O R P R O F S L O C O T O R P

55

Urine Concentration with Amicon Ultra Centrifugal Filters

n o i t a r t n e c n o C e n i r U • S N I E T O R P R O F S L O C O T O R P

56

Introduction

Materials

The measurement of specific proteins in urine is important for the diagnosis and management of disease states. In most cases, the content of these proteins in urine is too low to be detected and needs to be concentrated. Amicon Ultra-4 devices can be used to concentrate urine samples prior to clinical laboratory analyses. For example, patients with multiple myeloma exhibit a proliferation of one antibody-producing plasma cell, which leads to excess production of free immunoglobulin light chains known as Bence-Jones proteins. After sample enrichment in the Amicon Ultra-4 10K NMWL device, immunofixation electrophoresis can be used to identify free light chains (Bence-Jones proteins) in urine by forming a light chain-antibody complex. Also, agarose electrophoresis can be used to quantitate light chains and identify additional low molecular weight proteins such as albumin, a-1 globulins, transferrin and IgG that can be present in renal tubular disorders. Ultrafiltration of urine samples in Amicon Ultra-4 devices provides reproducible, high sample recovery for electrophoretic analyses, usually in 45 minutes or less.

• Amicon Ultra-4 device, 4 mL, 10K NMWL • Centrifuge with fixed-angle or swinging bucket rotor capable of 3400 x g • Kit for microprotein determination (i.e. Sigma cat. no. 610-A/Brilliant blue G/ Coomassie blue) • Pipetter with 200 µL tip • Electrophoresis (agarose gel) and immunofixation equipment with apparatus and reagents

Method 1. Determine the total protein in a 24-hour urine specimen. 2. Fill Amicon Ultra-4 device with 4 mL of urine. 3. Centrifuge at 3400 x g for 30–45 minutes (approximately 25–50 µL concentrate volume). This produces up to a 160-fold increase in concentration. 4. Insert a pipetter into the bottom of the filter unit and withdraw the concentrated sample. 5. Perform agarose electrophoresis on the concentrate to quantitate light chains and identify other proteins. Determine the percentage of light chains with respect to the total number of components in the urine. Then multiply the percentage of light chains by the total 24-hour protein concentration (grams per 24-hour volume). 6. Perform immunofixation electrophoresis on the concentrate to identify light chains.

Acknowledgements

Additional Notes

Research using Amicon Ultra devices for urine concentration in this protocol was conducted by Mark Merchant, Ph.D. at Helena Laboratories, Beaumont, TX.

1. Amicon Ultra devices can also be used to concentrate serum, plasma and cerebrospinal fluid for similar analyses. A concentration of approximately 20 mg/mL is required in order to detect free light chains from diseased patients by agarose electrophoresis. Detection by immunofixation electrophoresis is 10 times more sensitive than by agarose electrophoresis.

References 1. Tietz, N. Clinical Guide to Laboratory Tests, 2nd ed. Philadelphia: Saunders, WB; 1990;362–363. 2. Kahns L. Clinical Chemistry 1991;37: 1557–1558. 3. Cleveland Clinic homepage. Accessed July 2002. www.clevelandclinic.org/myeloma/ DiagnosisAndTreatmentOf MultipleMyeloma.html 4. Christenson RH, et al. Clinical Chemistry 1983;29(6):1028–1030. 5. Christenson RH, and Russell ME. Clinical Chemistry 1985;31(6):973.

2. Normal heterogeneous immunoglobulins may also be seen in urine concentrate with immunofixation electrophoresis. This “ladder effect” is comprised of microheterogenous light chains. Bence-Jones proteins may be within this ladder. To verify the presence of Bence-Jones proteins requires additional analysis by two-dimensional electrophoresis. 3. If there is excess antigen, dilution of the concentrate will be required until equilibrium is achieved between the antigen (Bence-Jones protein) and the antibody. 4. Millipore also offers static concentrators (Minicon® devices) for concentration of Bence-Jones protein in urine.

n o i t a r t n e c n o C e n i r U • S N I E T O R P R O F S L O C O T O R P

57

Use of Centrifugal Filter Devices as an Alternative to Stirred Cells

s l l e C d e r r i t S o t e v i t a n r e t l A n a s a s e c i v e D r e t l i F l a g u f i r t n e C f o e s U • S N I E T O R P R O F S L O C O T O R P

58

Introduction

Abstract

Stirred cell devices have been successfully used to purify proteins from large volumes of solution for many years. However, the need for assembly and cleaning between uses, and the lengthy separation times, can present difficulties for active, or understaffed, laboratories. Millipore’s large volume centrifugal filter devices come preassembled and ready to use. Individual devices are available for processing 20 or 70 mL volumes. For larger volumes, multiple devices can be spun simultaneously. Spin times are typically measured in minutes. And unlike stirred cells, centrifugal devices run unattended. There is no need to refill a reservoir and, therefore, less risk of adventitious contamination. The following protocol demonstrates the use of Centricon Plus-70 centrifugal filter devices to purify and concentrate proteins from large volumes of solution.

This study aims to achieve purification and concentration of a fusion protein composed of the alpha and gamma subunits of the human high affinity IgE receptor. The alpha-gamma fusion protein is purified and then coupled to Sepharose® (GE) beads to produce an affinity column for isolating human IgE antibodies.

Figure 1.

Method The alpha and gamma subunit components of the high affinity IgE receptor were transfected into mouse myeloma cell line NS0 and the secreted fusion protein was purified on a Protein G column. The eluted proteins were concentrated from volumes of 500–1000 mL to 5–10 mL using a Centricon Plus-70 centrifugal filter device in a swinging bucket rotor at 3000 x g for 10 minutes (60 mL per filter unit). Buffer exchange was also performed subsequently using the same units in tissue culture grade sodium azide-free PBS. The concentrated samples were then filter-sterilized under sterile conditions using a 0.2 µm pore filter.

140

Results

120

Spectrophotometric evaluation of alpha-gamma fusion protein recovery using Centricon Plus-70 devices showed a result of 18.8 mg/L of supernatant. HPLC trace analysis revealed the chromatogram shown in Figure 1.

s t i 100 n U e 80 c n a b r 60 o s b A 40

Acknowledgement

20 0 0

5

10

15

20

25

Time (min)

HPLC profile of alpha-gamma fusion component of the human Fc εRI

30

Protocol courtesy of GKT School of Biomedical Sciences, London.

Protocols for Nucleic Acids

Purification of DNA Sequencing Reactions . . . 60 Concentrating and Desalting DNA or RNA . . . 63 Preparing Samples for Forensics ID Analysis . . 66 Concentration of Genomic DNA for Forensic Analysis . . . . . . . . . . . . . . . . . . . . . . 67 Purification of PCR Products . . . . . . . . . . . . . . 68 Quantitative Recoveries of Nanogram Amounts of Nucleic Acids . . . . . . . . . . . . . . . 70 RNA Purification and Preparation of Fluorescent cDNA Probe from Human mRNA . 72 Purification of In Vitro Synthesized mRNA . . . . 76 Effect of Centrifugal Ultrafiltration on Large Fragment DNA Integrity . . . . . . . . . . . . 78 DNA Extraction from Agarose Gels . . . . . . . . 80 PCR Purification. . . . . . . . . . . . . . . . . . . . . . . 82 Enzyme Removal . . . . . . . . . . . . . . . . . . . . . . 84

S D I C A C I E L C U N R O F S L O C O T O R P

59

Use of Microcon 100 Centrifugal Filter Devices to Purify DNA Sequencing Reactions

s n o i t c a e R g n i c n e u q e S A N D f o n o i t a c i f i r u P

Introduction

Method

DNA sequencing reactions performed using ABI’s BigDye® terminator chemistries require the removal of unincorporated dye-terminators and excess salt prior to injection into a sequencer. The traditional method for purifying DNA sequencing reactions is ethanol precipitation—a labor intensive method requiring multiple spins, incubation, and drying that can take up to an hour. Care must also be taken to retain the resulting pellet. Microcon 100 devices can remove contaminating salts and unincorporated dye terminators from DNA sequencing reactions and deliver SEQ quality and read lengths equal to or better than ethanol precipitation in 10 minutes. As capillary-based DNA sequencers become more prevalent, the need for single sample purification of SEQ reactions also increases. The first protocol achieved a time savings of 50 minutes over the traditional ethanol precipitation method while a second protocol, which relies on less vigorous centrifugation, took approximately as long as ethanol precipitation but excelled in experiments in which it was desirable to read close to the primer.

DNA was isolated according to standard protocols and amplified for sequencing analysis on an ABI model 3700 DNA sequencer using polymerase reagents and ABI’s BigDye Terminator v. 3.1. Sample was purified using two experimental protocols. Results were assessed using electropherograms, Phred 20 read lengths, and estimation of dye blobs.

Equipment

Key parameters for DNA sequence reactions include quality, read length, sequencing accuracy, ability to read close to the primer, and the presence of dye blobs. The first three factors may be assessed using Phred 20 scoring, which is performed automatically via software. Phred 20 corresponds roughly to a 99% probability that a base call is accurate. Ability to read close to the primer is measured by the Primer + value, which relies on both software and visual verification. Dye blobs are artifacts of unknown origin, occurring during sequencing or cleanup, that may overshadow peaks. Dye blob assessment is typically performed by visually examining the electropherogram.

• S D I C A C I E L C U N R O F S L O C O T O R P

60

• Instrument: ABI sequencer cat. no.3700 • Reagent: ABI BigDye Terminator v3.1 • Injection parameters: existing parameters developed for Montage Injection solution. • Data output: Graphs and electropherograms that show A) removal of dye-terminators (blobs); B) Read length (Phred 20 scores); C) Primer

Microcon 100 protocol 1. Sequence Product (10 µL) + 100 µL of Montage injection solution in a Microcon 100 device 2. Spin for 2.5 minutes at 12,000 x g 3. Add 100 µL of injection solution to device 4. Spin for 2.5 minutes at 12,000 x g 5. Resuspend in 20 µL of injection solution then incubate for 2 minutes 6. Invert spin for 2 minutes at 1,000 x g 7. Inject into sequence analyzer The above steps were completed in 10 minutes— at least one third the time required for ethanol precipitation.

Results

Table 1. Results from a sequence purification using the Microcon 100 device Dye Terminator

Phred 20

Dye Blobs

Primer +

1/2X 1/8X

646 ± 59 656 ± 42

0.4 None observed

11 ± 10 8±7

n=48

Figure 1. Electropherogram of a 1/2X SEQ reaction, purified using the Microcon 100 protocol

s n o i t c a e R g n i c n e u q e S A N D

Microcon 100 at 12,000 x g

f o n o i t a c i f i r u P

Phred 20–684

Primer + = 8

Dye blobs = 0


61

Conclusion Microcon 100 ultrafiltration devices represent a rapid and efficient alternative to ethanol precipitation when preparing DNA for sequencing analysis. The high-spin protocol outlined above affords a significant reduction (10 minutes vs. 60 minutes) in sample preparation time. For experiments where low Prime + is required, researchers can employ a lowspeed centrifugation protocol that provides read lengths equal to or better than ethanol precipitation, with far less opportunity for user error.

Table 2. Results from a typical sequence purification using an alternate protocol Dye Terminator

Phred 20

Dye Blobs

Primer +

1/2X 1/8X

627 ± 83 653 ± 73

0.6 None observed

1±0 1.7 ± 3

Ethanol

605 ± 88

0.5

2.6 ± 4

Figure 2. Electropherogram of a 1/8X SEQ reaction, purified using an alternate protocol

s n o i t c a e R g n i c n e u q e S A N D f o n o i t a c i f i r u P • S D I C A C I E L C U N R O F S L O C O T O R P

62

Microcon 100 at 500 x g

Phred 20 = 622

Primer + = 1

Dye blobs = 0

Concentrating and Desalting DNA or RNA with Microcon and Amicon Ultra Centrifugal Filters Introduction Centrifugal filter devices serve as powerful tools in molecular biology applications, such as DNA or RNA concentration and desalting procedures. Ultrafiltration (UF) is a pressure-driven, convective process that uses semipermeable membranes to separate species by molecular size and shape. UF is highly efficient, allowing for concentration and desalting at the same time. Unlike the use of chemical precipitation methodologies (i.e., ethanol, phenol/chloroform), there is no phase change, which often denatures labile species. DNA and RNA samples with starting concentrations as low as 5 ng/mL can be routinely concentrated in minutes with 99% recovery of starting material, and without the use of co-precipitants. Centrifugal concentrator devices are ideal for separating high and lowmolecular weight species. Ultrafiltration can also be used to change solvents by diafiltration. In this process, the sample is concentrated, then diluted to the original volume with the desired buffer and concentrated again, thus “washing out” the original solvent.

Table 1. Nucleotide cut-off guidelines for Microcon centrifugal devices (based on >90% recovery of nucleic acids)

NMWL

Single-Stranded* Nucleotide Cut-off (bp)

Double-Stranded Nucleotide Cut-off (bp)

3K 10K 30K

10 30 60

10 20 50

50K 100K

125 300

100 125

*Single-stranded nucleic acids with extensive secondary structure will be better retained than those without.

Millipore ultrafiltration membranes are characterized with well-defined, globular solutes (proteins). Typically, the nominal molecular weight limit (NMWL) for a membrane is the point at which over 90% of a solute with that molecular weight will be retained. To process DNA or RNA, the membrane needs to be characterized according to the number of nucleotides in the fragment. Polynucleotides (DNA and RNA) have tertiary structures that are ordinarily more extended than those of typical globular proteins of similar size. Millipore has determined nucleotide cut-offs (based on the number of bases or base pairs in a fragment of DNA or RNA) that correspond to the NMWL of each of their low binding membranes. The nucleotide cut-off (NCO) indicates the fragment length of single- or double-stranded DNA or RNA that one would expect to recover at 90% efficiency with a unit of the named NCO. It is best to choose the NCO with about half the length of the fragment of interest. For example, selecting a 30K NMWL membrane for a 50 base pair fragment generally results in 90% product recovery. A 10K NMWL membrane would provide for closer to 100% recoveries but would take much longer to process the sample. For nucleic acid samples >500 base pairs, a 100K NMWL membrane is appropriate. Table 1 offers guidelines for DNA/RNA retention based on the nucleotide content of single- and double-stranded pieces. For example, more than 90% of a single-stranded 30-mer will typically be retained by a Microcon 10K NMWL. Concentrating dilute DNA solutions is a key step for many subsequent preparative and analytical procedures. For example, standard plasmid preparations involving cesium chloride, equilibrium centrifugation, and gel filtration yield DNA in large volumes that require concentrating prior to precipitation. DNA concentration is also necessary in purifying restriction fragments from gels.

A N R r o A N D g n i t l a s e D d n a g n i t a r t n e c n o C • S D I C A C I E L C U N R O F S L O C O T O R P

63

There are three major techniques currently available for concentrating nucleic acids: • repeated extractions with n-butanol • adsorption to ion exchange resin, followed by high salt elution • lyophilization The first method has the disadvantage that n-butanol concentrates all solutes, including salt, which tends to co-precipitate with DNA up on addition of ethanol. The second method, ion exchange, aside from requiring buffers of various ionic strengths, yields DNA in a high salt solution. The third method, lyophilization, increases the concentration of buffer components, which can result in degradation of nucleic acids. Ultrafiltration membranes retain DNA or RNA but are permeable by smaller ionic buffer components. Ultrafiltration alone does not change buffer compo A N R r o A N D g n i t l a s e D d n a g n i t a r t n e c n o C

Methods Table 2. Desalting and recovery with Microcon 30K NMWL devices

Microcon Device

Spin Number

% DNA Recovered

% CsCl Removed

1 2 3

88 89 88

99.9 100 100

1. Select a Microcon unit with nucleotide cut-off equal to or smaller than the molecular size of the nucleic acid you want to retain (refer to Table 1). 2. Insert Microcon sample reservoir into one of the two vials provided for each unit.

Using 1 mL of 25 µg/mL E. coli DNA in 6 M CsCl, sample was repeatedly concentrated to 0.1 mL. Sample spun at 2000 x g in a 45 degree fixed angle rotor. The concentrated sample was reconstituted to original 1 mL volume by adding 50 mm Tris. After three spins, CsCl concentration was reduced by four orders of magnitude.


sition. The salt concentration in a sample concentrated by Microcon centrifugal filter devices will be the same as in the original sample. For desalting, the concentrated sample is diluted with water or buffer to its original volume and spun again in a process called diafiltration. This removes the salt by the concentration factor of the ultrafiltration. For example, if a 500 µL sample containing 100 mM salt is concentrated to 25 µL (20X concentration factor), 95% of the total salt in the sample will be removed. The salt concentration in the sample will remain at 100 mM. Rediluting the sample to 500 µL will bring the salt concentration to 5 mM. Concentrating to 25 µL once more will remove 99% of the original total salt. The concentrated sample will now be in 5 mM salt. For more complete salt removal, an additional redilution and spinning cycle will remove 99.9% of the initial salt content (see Table 2).

Table 3. Recommended g-force and spin time for Microcon devices NMWL

Maximum G-Force Rating

Spin Time (min) at 4 °C


3K 10K 30K

14,000 14,000 14,000

185 50 15

95 35 8

50K 100K

14,000 500

10 25

6 15

3. To concentrate (without affecting salt concentration): Pipette up to 500 µL of DNA or RNA sample into the reservoir. Spin for recommended time, not exceeding recommended g-force guidelines shown in Table 3. 4. To exchange salt: Add the proper amount of appropriate the diluent to bring the concentrated sample to 500 µL. Spin for the recommended time, not exceeding g-force shown in Table 3. To achieve lower salt concentration, repeat the entire step as necessary. NOTE: Do not let filtrate vial overfill. 5. Remove reservoir from vial and invert into a new vial (save filtrate until sample has been analyzed). 6. Spin for 2 minutes at 500–1000 x g to recover nucleic acid in the vial. 7. Remove reservoir. Cap vial to store.

64

Amicon Ultra Device

Table 4. Nucleotide cut-off guidelines for Amicon Ultra-4

1. Select an Amicon Ultra unit with a nucleotide cut-off equal to or smaller than the molecular size of the nucleic acid you want to retain (refer to Table 4).

A. Nucleotide cut-off guidelines for single stranded DNA 3K (40 min)

10K (20 min)

10 15 20

98 96 94

97 95 94

25

96

96

2. To concentrate (without affecting salt concentration): Pipette up to 4 mL of DNA or RNA sample into the reservoir. Spin for the recommended time, not exceeding g-force shown in Table 5. 3. To exchange salt: Add the proper amount of the appropriate diluent to bring the concentrated sample to 4 mL. Spin for the recommended time, not exceeding g-force shown in the Table 5. To achieve lower salt concentration, repeat the entire step as necessary. NOTE: Do not let filtrate vial overfill.

NMWL

Single-Stranded Nucleotide Cut-off (bp)

B. Nucleotide cut-off guidelines for double stranded DNA NMWL

Double-Stranded Nucleotide Cut-off (bp)

30K

50K

137 301 657 1159

95 — — —

95 94 99 100

100K

— — — 97

Based on >90% retention. Spinning 50 and 100K devices at higher than recommended g-force may result in lower recovery of DNA.

Table 5. Recommended g-force and spin time for Amicon Ultra devices NMWL

Maximum G-Force Rating


3K 10K

7,500 7,500

40 20

30K 50K 100K

5,000 2,000 2,000

10 20 10

A N R r o A N D g n i t l a s e D d n a g n i t a r t n e c n o C • S D I C A C I E L C U N R O F S L O C O T O R P

65

Preparing Samples for Forensics Identification Analysis Using Microcon Centrifugal Devices

s i s y l a n A n o i t a c i f i t n e d I s c i s n e r o F r o f s e l p m a S g n i r a p e r P

Centrifugal filter devices with ultrafiltration (UF) membranes are used by forensics laboratories to isolate genomic DNA for human identification. The source materials are typically blood stains and/or other bodily fluids obtained from crime scenes, criminal suspects, or human remains. The isolated genomic DNA is used to identify individual persons based on their patterns of Short Tandem Repeats. STRs are polymorphic DNA loci that contain repeated DNA sequences. Because the repeats can vary from two to seven bases in length, many different alleles are possible for each locus. The parallel analysis of 9 to 13 polymorphic STR loci can unequivocally identify an individual.

Figure 1. A


66

B

Genotype analysis of a semen sample before and after concentration by a Microcon centrifugal filter. Electrophero- gram A shows a partial profile with a high molecular weight loci in each color missing. Electropherogram B shows a full profile and higher peak heights. Amounts amplified were 0.62 ng for electropherogram A and 1 ng for electropherogram B. The sample was extracted with Chelex beads. The peaks are labeled with allele name, size in base pairs, and fluorescent peak height. The improvement in results is due not only to the increased DNA input but also to the concentration step following extraction.

Several suppliers offer STR assay kits. Each supplier offers several versions of their kits with different protocols, modes of operation, and detection systems to suit the user’s needs. Millipore’s UF-based centrifugal devices are specified in several of these protocols (AmpFLSTR® Profiler, Applied Biosystems; GenePrint® STR and GenePrint Fluorescent STR Systems, Promega).

Use of UF-based Centrifugal Devices in New York City Depending on the type of evidence under investigation, the Department of Forensic Biology in New York City had been preparing genomic DNA by either (1) organic extraction (phenol/chloroform/ isoamylalcohol) followed by alcohol precipitation or (2) direct lysis in the presence of Chelex® beads (Bio-Rad) with no further purification. The Department of Forensic Biology’s laboratory has replaced the ethanol precipitation step with Microcon centrifugal filters with a 100K NMWL ultrafiltration membrane. The centrifugal devices provide a faster way to concentrate the purified DNA without the use of toxic chemicals. The laboratory also uses the Microcon 100K NMWL device after Chelex extraction to remove inhibitors that can adversely affect PCR amplification. In addition, Chelex extraction of small bloodstain samples often results in DNA concentrations below detection limits. Concentrating extracted samples with Microcon 100K NMWL devices improves the success rate of STR amplification and yields a full STR allele profile even with m inute blood or semen stains. According to the New York City Department of Forensic Biology, several of the peaks shown in Figure 1 would have been below the detection threshold and identification would have been negative or inconclusive without sample cleanup using Microcon devices.

Concentration of Genomic DNA for Forensic Analysis Using Amicon Ultra-4 50K Centrifugal Devices Introduction Millipore’s Amicon Ultra-4 centrifugal filter devices provide fast ultrafiltration, with the capability for high concentration factors. The device design incorporates the Millipore Ultracel low binding regenerated cellulose membrane, providing excellent sample recoveries from dilute and complex sample matrices. The Amicon Ultra device is configured in a vertical design, which minimizes solute polarization and subsequent fouling of the membrane. The vertical design and available surface area provide for fast sample processing, high retentate recovery (typically greater than 90% of dilute protein or DNA concentrate), and the capability for very high concentration factors (>80-fold). The Amicon Ultra-4 devices are suitable for use with dilute nucleic acid samples which can be quickly ultrafiltered, allowing for fast separation of genomic DNA from low molecular weight (MW) compounds. The Amicon Ultra device design is compatible with the multiple spin recovery assays used for the purification of genomic DNA for forensic analysis. The concentrate is collected from the filter unit sample reservoir using a pipetter, while the ultrafiltrate can be collected in the provided centrifuge tube if desired. The filter device can be spun in a swinging bucket or a fixed angle rotor. The Amicon Ultra devices come fully assembled in a centrifuge tube, ready for sample use.

Protocol Recommendations for Genomic DNA Isolation • Dilute sample to 2.0 mL with TE Buffer (10 mM Tris, pH 7.6, 1 mM EDTA) • Centrifuge at 2,000 x g for 20 minutes at room temperature • Repeat for the number of washes desired Note: The centrifugation time is based on samples containing up to 2 μg of genomic DNA. For forensics samples, users will likely need to determine the appropriate centrifugation time as many factors (e.g., DNA concentration and temperature) will affect centrifugation time.

“ Amicon Ultra has enabled our testing lab to process samples quicker and with a higher convenience and confidence as the decreased number of handling steps allows us to be free of cross-contamination.” Professor Miguel Paredes, Barcelona Laboratory of Instituto Nacional de Toxicologia y Ciencias Forenses

s i s y l a n A c i s n e r o F r o f A N D c i m o n e G f o n o i t a r t n e c n o C • S D I C A C I E L C U N R O F S L O C O T O R P

67

Purification of PCR Products with an Amicon Ultra Device Introduction

s t c u d o r P R C P f o n o i t a c i f i r u P • S D I C A C I E L C U N R O F S L O C O T O R P

To enable the use of DNA fragments generated by the Polymerase Chain Reaction (PCR) in downstream applications, unincorporated primers, nucleotides and contaminating salts must to be quantitatively removed. The application of ultrafiltration to PCR clean-up is well established and represents a convenient and efficient approach to generate concentrated, purified PCR products suitable for such downstream applications such as cloning or DNA sequencing. Millipore offers a number of products designed for the purification of PCR products in 96-well and 384-well plate and 1-up formats. The protocol and data shown here demonstrate the utility of Amicon Ultra-4 devices for the purification of PCR products. By extension, these data can be used to select an Amicon Ultra with the appropriate molecular weight cut-off (MWCO) for use in the purification of DNA used in other applications.

Considerations For applications involving nucleic acids, strand length is the most useful parameter for selecting the Amicon Ultra device appropriate for a particular application. However other parameters including, DNA concentration, the magnitude of the driving force (g-force) and the salt concentration all act in concert to affect DNA recovery. When purifying PCR reactions for example, optimal yield can be

Figure 1. PCR recovery with Amicon Ultra-4 50K device

137

301

657

PCR Fragmentation Size (bp)

68

100 y r 80 e v o c e 60 R R C 40 P %

20 0

2,000 x g

5,000 x g

7,500 x g

achieved via dilution of the starting material and running at a relatively low g-force. For PCR fragments, an Amicon Ultra-4 device with a 50K MWCO membrane generally strikes the best balance between speed and recovery. If the DNA sample is in a buffer containing high salt and cannot be diluted, or the device is run at a higher than recommended g-force, a significantly reduced DNA recovery will likely be observed. Under these conditions, higher DNA or RNA recovery can be achieved by using the Amicon Ultra with the next tighter membrane, albeit with a much longer processing time. Note that, for maximal removal of unincorporated single-stranded primers from doublestranded DNA fragments, the molecular weights of the primer and DNA fragment should differ by at least an order of magnitude.

Method

) 100 e g n e 80 l l a h C 60 f o % ( 40 y r e v 20 o c e R 0

(n=8)

Figure 2. Effect of MWCO and g-force on the recovery of a 137 bp PCR product using Amicon Ultra-4 100K device

1159

DNA fragments (137, 301, 657 and 1159 bp) were generated by PCR using plasmid DNA as the template. After pooling to minimize deviceto-device variability, 100 µL aliquots of the PCR reactions were diluted with 2.0 mL of TE Buffer (10 mM Tris-HCL, pH 8.0, 1 mM EDTA) and added to an Amicon Ultra-4 device with a 50K MWCO membrane. The samples were centrifuged at 2,000 x g for 20 minutes, an additional 2.0 mL of TE buffer was added to the sampled and the PCR products were concentrated by a second 20-minute

spin at 2,000 x g. The purified PCR products were collected by pipette and the recovered DNA quantified by a fluorescent SYBR® Green I assay (Molecular Probes). The final sample volume was 87 ± 15 µL. The recoveries shown in Figure 1 demonstrate the high recovery that can be obtained from PCR reactions using this approach. The recovery of PCR fragments purified by ultrafiltration can be challenging when the resulting DNA fragments are <1000 bp in length. The amount of DNA recovered after purification can be maximized by using an Amicon Ultra device with a lower MWCO membrane (e.g., 50K versus 100K MWCO) and by centrifuging the devices at 2,000 x g as shown in Figure 2 for a 137 bp PCR fragment. Although increasing the g-force will speed processing time, it will do so at the expense of DNA recovery. For larger DNA’s, the effect is less dramatic (data not shown). The quantitative removal of single-stranded DNA primers or oligonucleotides from PCR reactions is critical for downstream applications. In order to demonstrate the ability of Amicon Ultra devices to efficiently remove primers, unpurified PCR products were spiked with 20 pmoles of a DNA reporter primer that had been labeled with fluorescein on its 5’ end. This primer (TCAG)5 was designed such that it had minimal secondary structure. After purification of the PCR products with an Amicon Ultra-4 device, the amount of primer remaining in the sample was determined by measuring fluorescence against a standard curve. The average primer removal in this experiment was 97.7 ± 1.4%. This level of primer removal has been shown to be sufficient for DNA sequencing and other important PCR-based applications.

Ultrafiltration-based purification relies on size exclusion to affect the separation of single stranded primers from the double stranded PCR fragments. As discussed above, the major challenge to optimizing ultrafiltration for PCR purification is with small, <1000 bp PCR pro ducts. Similarly, the efficiency of primer removal becomes more challenging as the size of the single stranded primer increases. As shown in Figure 4, the ability of an Amicon Ultra device with 30K MWCO membrane to remove primers is significantly diminished as the primer length approaches 50 bases. In contrast, when a 50K MWCO membrane is used, primer removal remains high and is consistent across the range of PCR primers evaluated. Whereas the use of a 100K would be expected to quantitatively remove all but the largest of primers, purification of small (e.g., <300 bp) PCR products is suboptimal when using a 100k MWCO membrane. Therefore, an Amicon Ultra-4 device with a 50K MWCO membrane provides the best balance of primer removal and recovery when used for PCR purification.

Conclusions The data presented herein demonstrate the utility of Amicon Ultra-4 devices for the purification of PCR products. By using devices with a 50K MWCO, users can expect to routinely obtain both high recoveries and efficient primer removal that will facilitate downstream applications. Although the analysis performed for this study focused on PCR purification, the general principles are applicable to purification of other nucleic acid preparations.

Figure 4. Effect of MWCO on primer removal in Amicon Ultra devices Figure 3. Amicon Ultra device efficiently removes unincorporated PCR primers ) 100 e g n e l l 80 a h C 60 f o % ( 40 l a v o 20 m e R 0

) e100 g n e l l 80 a h C f 60 o % ( 40 l a v o 20 m e R

0

20

25

s t c u d o r P R C P f o n o i t a c i f i r u P • S D I C A C I E L C U N R O F S L O C O T O R P

48

Primer Length (Bases) 137

301

657

1159

Amicon Ultra, 50k

Amicon Ultra, 30k

PCR Fragmentation Size (bp)

69

Quantitative Recoveries of Nanogram Amounts of Nucleic Acids with Microcon Centrifugal Filters Introduction

s d i c A c i e l c u N f o s t n u o m A m a r g o n a N f o s e i r e v o c e R e v i t a t i t n a u Q • S D I C A C I E L C U N R O F S L O C O T O R P

70

Molecular cloning experiments often require concentration of nanogram quantities of DNA. When dealing with such small amounts of nucleic acids, the use of coprecipitants (tRNA or purified glycogen) is essential for effective DNA recovery 1. However, when carrier tRNA cannot be used (for example, when DNA is to be labeled with 32P dATP and polynucleotide kinase), the only method of recovering DNA as a precipitate in ethanol has been the ultracentrifugation method developed by Shapiro2. Ultrafiltration, which has been traditionally used to concentrate and desalt protein samples simultaneously, can be an efficient alternative to ethanol precipitation. In this experiment, three popular ethanol precipitation protocols for DNA and oligonucleotides are compared with ultrafiltration in Microcon centrifugal filter devices. For a detailed discussion on the effects of incubation time, temperature and centrifugation parameters on ethanol precipitation, see Reference 3.

Methods Plasmid pBR322 was digested with EcoR I and the 3’ recessed termini were filled in with alpha 32P dATP, using Klenow fragment of DNA polymerase I. A 25-nucleotide mixed base oligomer was radiolabeled by phosphorylation with bacteriophage T4 polynucleotide kinase.

Ethanol Precipitation All DNA precipitations were performed in 250 µL volume. Each tube contained a given amount of DNA, supplemented with 10 ng of radioactively labeled pBR322 in 0.3 M sodium acetate buffer. To precipitate the DNA, 2.5 volumes of 95% EtOH were added to each tube. The content was well mixed and incubated for the specified period of time at –20 °C or –70 °C (dry ice). The solutions

were then centrifuged at 12,000 x g in a fixedangle microcentrifuge at 4 °C for 15 minutes. The supernatants were carefully removed and pellets resuspended in 50 µL of TE buffer. The radioactivity in the precipitates and supernatants was then determined by counting Cherenkov radiation. The percentage of recovered DNA was calculated from precipitates and the initial counts. Each data point in the tables represents the average of at least three samples. To precipitate the oligomer, 60 µL of TE buffer containing a given amount of 25-mer and 5 ng of radiolabeled tracer was supplemented with 240 µL of 5 M ammonium acetate and 750 µL of EtOH. Subsequent steps were identical to those described for DNA.

Ultrafiltration A solution of DNA (500 µL) was placed into a Microcon 30K NMWL unit and spun at 12,000 x g for 10 minutes. A 500 µL solution of oligonucleotides in TE buffer was placed into a Microcon 3K NMWL unit and spun for 45 minutes at 12,000 x g. The retentates were recovered by inverting the units and centrifuging at 500–1000 x g for 2 minutes. While concentrating the oligomer samples, it is important to avoid high salt concentrations which promote binding of single-stranded nucleic acids to the cellulose-based ultrafiltration membrane. The radioactivity in the retentates and filtrates was determined by counting Cherenkov radiation. Data represent averages of six samples each.

Results Standard methods for ethanol precipitation of nucleic acids and ultrafiltration are compared in Tables 1 and 2. Recovery of DNA was assessed at close to 100%, indicating that no significant amount of nucleic acid was lost to the membrane or device due to adsorption.

In contrast, DNA recovery using EtOH precipitation varied from as little as 14% as in the case of DNA (10 ng/mL) precipitated for 15 minutes at –70 °C to a maximum of 76% after overnight incubation at –20 °C.

Discussion Poor recovery of nucleic acids at very low concentration with ethanol precipitation may be partially due to the fact that small amounts of DNA do not adhere well to the tube walls following sedimentation unless high g forces (ultracentrifugation) are employed. The yield of DNA incubated at – 70 °C is slightly reduced, in agreement with previous studies. Also an overnight precipitation at – 20 °C can significantly improve recovery. The ultrafiltration devices were found to concentrate and desalt nucleic acids effectively in one step resulting in high recoveries and providing a quick, alternative to ethanol precipitation.

References 1. Wallace DM. Precipitation of Nucleic Acids. Methods in Enzymology 1987;152:41–8. 2. Shapiro DJ. Quantitative Ethanol Precipitation of Nanogram Quantities of DNA and RNA. Anal Biochem 1981;110:229–31. 3. Crouse J, Amorese D. Ethanol Precipitation: Ammonium Acetate as an Alternative to Sodium Acetate. Focus 1987;9(2):3–5.

s d i c A c i e l c u N f o s t n u o m A

Table 1. Efficiency of different methods for concentration of DNA DNA Concentration 10 ng/mL % Recovery

25 ng/mL % Recovery

50 ng/mL % Recovery

250 ng/mL % Recovery


EtOH, –70 °C, 15 min

14

15

23

52

55

EtOH, –20 °C, 30 min EtOH, –20 °C, 18 hrs

13 31

20 45

25 45

60 76

72 67

Microcon 30K NMWL Device

95

95

98

98

99

Method

Table 2. Efficiency of different methods for concentration of oligonucleotides

•

Oligomer Concentration 10 ng/mL % Recovery

25 ng/mL % Recovery

50 ng/mL % Recovery



EtOH, –70 °C, 15 min EtOH, –20 °C, 30 min EtOH, –20 °C, 18 hrs

4 6 17

4 5 20

5 6 15

6 4 30

33 33 58

Microcon 30K NMWL Device

93

94

93

95

95

Method

m a r g o n a N f o s e i r e v o c e R e v i t a t i t n a u Q S D I C A C I E L C U N R O F S L O C O T O R P

71

RNA Purification and Preparation of Fluorescent cDNA Probe from Human mRNA with Microcon Centrifugal Filters A N R m n a m u H m o r f e b o r P A N D c t n e c s e r o u l F f o n o i t a r a p e r P d n a n o i t a c i f i r u P A N R • S D I C A C I E L C U N R O F S L O C O T O R P

72

Introduction When working with RNA, introduction of RNAse contamination during sample preparation is of major concern. Sample yield and integrity can impact the efficiency of subsequent translations, hybridizations, protein binding, antisense, or ribozyme studies. A series of experiments was performed to determine the effectiveness of using Amicon centrifugal ultrafiltration devices from Millipore to concentrate and diafilter RNA samples. The results indicate remarkably high RNA recoveries and low adsorption losses, especially with prior membrane passivation. Microarrays are efficient tools that enable the high throughput identification of genes that are differentially regulated in response to disease, drugs or other stimuli. With the completion of several key genome sequencing projects, scientists now have the ability to custom design DNA microarrays specific to their research interests. The recent advances in robotics, bioinformatics and detection technologies have greatly simplified the manufacture and analysis of microarrays. However, the successful application of microarray technology requires highly purified, fluorescently labeled cDNA probes. The application of ultrafiltration technology to this challenge has resulted in a robust, efficient and rapid method for the genera-

tion of high quality fluorescently labeled probes suitable for use with microarrays. The protocol below describes a method for the generation and purification of fluorescently labeled probes using a Microcon 30K NMWL centrifugal filter device.

RNA Sample Integrity The primary objective of the study was to determine the effect of centrifugation on RNA integrity. The second objective was to determine any gross changes in the RNA that could be a result of contact of the sample with the ultrafiltration devices themselves. An RNA transcript was made, using MEGAscript® T7 RNA polymerase (Ambion) and DNA template Riboprobe® Gemini (Promega) according to manufacturer’s protocols. This transcript served as the starting RNA sample for the experiments discussed below. Figure 1 is an autoradiogram of labeled RNA. It compares an untreated sample to samples that were concentrated in Microcon 100K NMWL and 30K NMWL devices, used directly as supplied. The Microcon 100K NMWL unit was spun at a force of 3,000 x g and the Microcon 30K NMWL unit at 12,000 x g. As is evident from the autoradiogram, both unfiltered samples are virtually indistinguishable from the starting material.

Figure 1. Concentration of RNA transcript. RNA is intact and recovered with high efficiency.

Lane 1: Starting material Lane 2: RNA concentrated in a Microcon 100K NMWL device Lane 3: RNA concentrated in a Microcon 30K NMWL device

1 1.4 kb

0.68 kb

2

3

Method

7. Neutralize by addition of 15 µL of 0.1 N HCl.

Generation and Purification of Fluorescently Labeled Probes

8. Add 380 µL of TE (10 mM Tris, 1 mM EDTA) to a Microcon 30K NMWL device column. Next add the 60 µL of Cy5 probe and the 60 µL of Cy3 probe to the same Microcon device.

1. To anneal primer, mix 2 µg of mRNA or 50–100 µg total RNA with 4 µg of a regular or anchored oligo-dT primer in a total volume of 15.4 µL as shown in Table 1.

NOTE: If re-purification of Cy dye flow-through is desired, do not combine probes until Wash 2.

2. Heat to 65 °C for 10 minutes and cool on ice. 3. Add 14.6 µL of reaction mixture each to Cy3 and Cy5 reactions as shown in Table 2. 4. Incubate at 42 °C for 1 hour. 5. Add 1 µL SSII (RT booster) to each sample. Incubate for an additional 0.5 –1 hours.

A N R m n a m u H

9. Wash 1: Spin column for 7 to 8 minutes at 14,000 x g. 10. Wash 2: Remove flow-through and add 450 µL TE and spin for 7–8 minutes at 14,000 x g. It is a good idea to save the flowthrough for each set of reactions in a separate microcentrifuge tube.

6. Degrade RNA and stop reaction by addition 15 µL of 0.1 N NaOH, 2 mM EDTA and incubate at 65–70 °C for 10 minutes. If starting with total RNA, degrade for 30 minutes instead of 10 minutes.

Table 1. Preparation of primer Primer

Cy3 (µL)

Cy5 (µL)

mRNA (1 µg/µL) Oligo-dT (4 µg/µL)

x 1

y 1

Purified H2O (DEPC)

to 15.4

to 15.4

—

15.4

15.4

—

Total volume

Notes

2 µg of each if mRNA; 50–100 µg if total RNA Anchored: 5’-T T T T T T T T T T T T T T T T T T T T V N-3’

Table 2. Preparation of probe Reaction Mixture

Volume (µL)

Unlabeled dNTPs

Volume (µL)

Final Concentration (mM)

5X first-strand buffer*

6.0

dATP (100 mM)

25

25

0.1 M DTT

3.0

dCTP (100 mM)

25

25

Unlabeled dNTPs Cy3 or Cy5 (1 mM, GE Healthcare) Superscript® II (200 U/µL, Gibco-BRL)

0.6

dGTP (100 mM)

25

25

3.0

dTTP (100 mM)

10

10

2.0

Purified H2O

15

—

Total volume

14.6

Total volume

100

—

*5X first-strand buffer: 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl 2

m o r f e b o r P A N D c t n e c s e r o u l F f o n o i t a r a p e r P d n a n o i t a c i f i r u P A N R • S D I C A C I E L C U N R O F S L O C O T O R P

73

11. Wash 3: Remove flow-through and add 450 µL 1X TE, 20 µg of Cot1 human DNA (20 µg/µL, Gibco-BRL), 20 µg polyA RNA (10 µg/µL, Sigma cat. no. P9403) and 20 µg tRNA (10 µg/µL, Gibco-BRL cat. no. 15401-011). Spin 7–10 minutes at 14,000 x g. Look for concentration of the probe in the Microcon device. The probe usually has a purple color at this point. Concentrate to a volume of less than or equal to the volume listed in the “Probe and TE” column in Table 3. These low volumes are attained after the center of the membrane is dry and the probe forms a ring of liquid at the edges of the membrane. Do not dry the membrane completely.

A N R m n a m u H m o r f e b o r P A N D c t n e c s e r o u l F f o n o i t a r a p e r P d n a n o i t a c i f i r u P A N R

12. Invert the Microcon device into a clean tube and spin briefly at 14,000 RPM to recover the probe. 13. Select the appropriate row from Table 3. Adjust the probe volume to the value indicated in the “Probe and TE” column. 14. For final probe preparation add 4.25 µL 20X SSC and 0.75 µL 10% SDS. When adding the SDS, be sure to wipe the pipette tip with clean, gloved fingers to remove excess SDS. Avoid introducing bubbles and never vortex after adding SDS. The probe is ready for hybridization.

Results RNA recovery is a function of the initial RNA concentration and the buffer salt concentration. Figure 2 shows RNA transcript recovery in Microcon 100K. NMWL unit as a function of RNA concentration. For all concentrations evaluated, retentate recovery is above 85%, even at an initial concentration as low as 25 ng/mL. Figure 2 also displays the material monitored in the filtrate and on the membranes of the Microcon units. (Membranes were removed and counted without further treatment.) The amount of RNA in the filtrate does not vary significantly as a function of concentration. The RNA recovered on the membranes varies from <1% at high initial concentrations to 4% at the lowest concentration tested. Millipore’s Amicon centrifugal ultrafiltration devices contain low-binding cellulosic Ult racel-YM membranes. Nitrocellulose is commonly used to immobilize RNA, generally in high salt concentrations. Figure 3 shows RNA recovery as a function of buffer salt concentration. The data show that both retentate and total recoveries fall as salt concentration increases. An increase in the amount of material on the membrane is also seen (from 2% to 6%) as the salt concentration increases from 10 to 500 mM. Similar trends are seen in the use of Microcon

Table 3. Final probe preparation Cover Slip Size

Total Hybrid Volume (µL)

Probe and TE (µL)

20X SSC* (µL)

10% SDS (µL)

•

22 x 22

15

12

2.55

0.45


22 x 40

25

20

4.25

0.75

22 x 60

35

28

5.95

1.05

74

*20X SSC: 3.0 M NaCl, 300 mM NaCitrate (pH 7.0)

100K NMWL devices, except that retentate recoveries fall to 60% at 500 mM salt. It was possible to count the devices themselves in addition to the filtrate and membrane. The results show a slight increase in counts on the membrane with increasing salt concentration. Significantly more (from 9% to 17%) material remained on the device as salt concentration increased. Passivation increased RNA recovery to 80%, regardless of initial salt concentration. This is attributed to a decrease in the amount of RNA adhering to the device. When the devices were passivated, RNA loss due to adsorption was reduced to 2%, regardless of salt concentration.

Conclusion The results shown here demonstrate that RNA samples can be concentrated or diafiltered with Microcon concentrators without loss of RNA integrity. RNA recovery in the retentate is typically >85%. As the salt concentration of the buffer system increases, RNA retentate recovery decreases, principally due to RNA adhering to the device rather than to the membrane. RNA recovery from solutions with high initial salt concentrations can be significantly improved by pre-treating the devices with a 5% SDS solution.

Acknowledgements Fluorescent probe preparation protocol courtesy of Patrick Brown, Max Diehn, and Ash Alizadeh, Stanford University School of Medicine.

Figure 2. 100%

80%

d e r e 60% v o c e R A 40% N R 20%

0% 0.025

0.25

0.5

1.0

5.0

10.0

RNA Concentration (µg/mL) Microcon Filtrate

Microcon Membrane

Microcon Retentate

RNA recovery in Microcon concentrators. RNA was concentrated as described in text. Cherenkov counts of Microcon retentate, filtrate and membrane (n=3 units) were compared to total starting counts.

Figure 3. 100%

80%

d e r e 60% v o c e R A 40% N R 20%

0% 10

100

200

500

NaCl Concentration (mM) Filtrate

Membrane

Device

Retentate

Improvement in RNA recoveries using Microcon devices (n=6) which were pre-treated with a 5% SDS solution.

A N R m n a m u H m o r f e b o r P A N D c t n e c s e r o u l F f o n o i t a r a p e r P d n a n o i t a c i f i r u P A N R • S D I C A C I E L C U N R O F S L O C O T O R P

75

Purification of In Vitro Synthesized mRNA with Microcon Centrifugal Filters

A N R m d e z i s e h t n y S o r t i V n I



76

Introduction

Methods

In vitro transcription reactions employing T3, T7 or SP6 phage-encoded RNA polymerases are widely used to synthesize RNA from recombinant vectors containing appropriate promoters. Production of large amounts of specific RNA is valuable in the preparation of hybridization probes and in vitro translation studies; in the synthesis of ribozymes, rRNA, SRP, antisense RNA and substrates for RNA splicing; and in RNA-protein interaction studies. Microcon centrifugal filters are well suited for the purification of radiolabeled RNA transcripts1. Ultrafiltration can simultaneously and efficiently remove unincorporated ribonucleotides and salts from the transcripts and concentrate the RNA. RNA molecules retain their integrity and are recovered with high yields. Purity of a transcript is especially important when it is used in in vitro translation systems. Trace amounts of ethanol, phenol, salts or excess cap analog used during the synthesis of capped mRNA can cause a dramatic decrease in translation efficiency. After the transcription reaction is complete, template DNA is usually degraded by the addition of DNase I. The RNA is purified by two phenol/ chloroform extractions followed by ethanol precipitation. Other, less popular methods are gel purification (used predominantly when separation of full-length transcript from shorter RNAs is important, e.g., ribonuclease protection assays) or LiCl precipitation. A series of experiments was performed in our laboratory to determine the effectiveness of using Microcon devices to purify in vitro synthesized mRNA and in vitro translation studies. Results indicate that ultrafiltration can efficiently remove inhibitory contaminants from mRNA preparations, leading to increased translational efficiencies.

RNA Transcription For our studies we chose plasmid pGEM-luc containing the luciferase gene (luc) in the center of a multiple cloning cassette of the pGEM-11Zf (-) plasmid (Promega). DNA template was linearized with XhoI, followed by enzyme and salt removal by diafiltration in Microcon 100K NMWL devices. Linearized template was transcribed, using MEGAscript® kit (Ambion) according to the recommended protocol. After the reaction was completed (3 to 4 hours), template DNA was degraded with DNase I and the reaction mix added to a Microcon 30K NMWL device filled with 450 µL of water. The device was spun for 20 minutes at 12,000 x g in a temperature-controlled centrifuge at 4 °C. Purified, concentrated RNA was recovered by inverted spin. For the preparation of capped transcript, cap analog m7G (5’) ppp (5’) G (New England Biolabs, Inc.) was included in the transcription reaction and the level of GTP reduced (4:1 ratio of cap analog to GTP). To purify the transcript by phenol/chloroform extraction, the reaction mix was diluted with water and a one-tenth volume of ammonium acetate stop solution was added. The mixture was extracted once with phenol/chloroform, followed by chloroform extraction. RNA was precipitated with isopropanol and the pellet resuspended in distilled water. Alternatively, LiCl precipitation solution (one-half volume) was added to the reaction mix, followed by incubation at minus 20 °C for 1 hour. RNA was pelleted by centrifugation and dissolved in water. Size and integrity of the in vitro transcription products were assessed by running an aliquot of the purified RNA transcript on a formaldehyde/formamide agarose gel. Ethidium bromide was added to the RNA before lading on the gel to stain the RNA sample and keep background fluorescence low2.

Translation

In Vitro

In vitro translations were performed in the Flexi™ Rabbit Reticulate Lysate System (Promega) according to standard luciferase RNA translation conditions with minor modifications (Rnasin Ribonuclease inhibitor was omitted and 35S-methionine added). Results of translation were analyzed by determination of percent incorporation of 35S-methionine and fold stimulation, compared to controls without RNA. Minimum acceptable stimulation was 8-fold.

Results Aliquots of RNA transcript purified by different methods (ultrafiltration, phenol extraction and LiCl precipitation) were run on a denaturing agarose/ formaldehyde gel. Results are shown in Figure 1. The banding pattern of the 1.7 kb RNA transcripts is identical regardless of purification method. Similar results were obtained in the case of capped transcript (results not shown). The effect of increasing the mRNA concentration on the translational efficiencies was examined. At low mRNA levels, the capped luc mRNA was translated three times more efficiently than the uncapped mRNA (Figure 2). At higher mRNA levels, the translation of both transcripts was comparable. Similar behavior was observed with CAT mRNA3. Even relatively high levels of mRNA did not cause the decrease in translational efficiencies noted by other groups4. This result could be attributed in part to the lack of inhibitory contaminants in the mRNA preparation. We also checked the effect of the RNA clean-up method on in vitro translational efficiency. For details of various procedures, see the Methods section. RNA purified by each of the methods (1 µg) was translated and analyzed. While there were no observable differences between these RNAs by gel electrophoresis analysis (Figure 1), RNA purified by ultrafiltration gave twice the translation efficiencies of phenol-extracted RNA.

References 1. Krowczynska AM. BioSolutions 1993;2(1):1–2. 2. Ogretmen B, Ratajczak H, Kats A, Stark BC. Biotechniques 1993;14(6):932-5. 3. Polayes D. Focus 1991;13(4):130–2. 4. Dasso MC, Jackson RJ. Nucl Acid Res 1989; 17:3129.

Figure 1.

1

2

3

4

Comparison of pGem-luc transcript purification methods. Transcripts were synthesized in 20 µ L reactions. After DNase I treatment, RNA was purified from the reaction mix. 500 ng of purified RNA were run on 1% agarose/ formaldehyde gel.

Lane 1: 0.16–1.77 kb RNA ladder (Gibco BRL) Lane 2: RNA purified in Microcon 30K NMWL device Lane 3: RNA purified by phenol extraction Lane 4: RNA purified by LiCl precipitation

A N R m d e z i s e h t n y S

Figure 2. 140

o r t i V

120 100

n I

4

0 1 80 x m 60 p c


Capped pGem-luc RNA pGem-luc RNA

40 20 0

•

0

0.5

1.0

1.5

2.0

µg RNA/50 µL Reaction Effect of pGem-luc RNA concentration on in vitro translation. Increasing amounts of uncapped Luc RNA transcripts and capped Luc RNA were used in translation reactions. Incorporation of 35 S-methionine was determined by TCA precipitation. Both RNA transcripts were purified with M icrocon 30K NMWL devices.


77

Effect of Centrifugal Ultrafiltration on Large Fragment DNA Integrity Introduction

y t i r g e t n I A N D t n e m g a r F e g r a L n o n o i t a r t l i f a r t l U l a g u f i r t n e C f o t c e f f E • S D I s C d A i c C A I E c i L e C l c U u N N R O • F S L O C O T O R P

78

Centrifugal ultrafiltration provides a fast and easy method for the concentration and desalting of biological molecules. Previous reports document the use of ultrafiltration for desalting samples of nucleic acids1, removing excess primers from PCR2 reactions3, or concentrating DNA/RNA samples without the need to use ethanol precipitation4. Microcon filters offer a convenient, reproducible means for centrifugal ultrafiltration of samples from 50 µL to 2 mL. They insure high sample recovery with their patented inverted recovery spin. The analysis of complex genomes depends on the ability of the researcher to prepare pure, high molecular weight DNA. When preparing high molecular weight DNA samples for cosmid cloning or other applications, it is important to treat the DNA gently to avoid shearing or other damage to the sample. As virtually all protocols for the preparation of high molecular weight DNA require, at some point, buffer exchange and/or concentration, centrifugal ultrafiltration can provide an efficient alternative to standard procedures. To be useful, centrifugation must not cause breakage of the DNA during the ultrafiltration procedure. This article summarizes the results of a series of experiments designed to evaluate the effect of g forces on various samples of high molecular weight DNA during centrifugal ultrafiltration in Microcon units.

Methods DNA Ladder As a model system to check for the introduction of single-strand nicks during the ultrafiltration spin, supercoiled Ladder DNA (Gibco-BRL, Gaithersburg, MD) which ranges in size from 2 to 8 kb was used. DNA diluted with TE buffer was loaded into Microcon 30K NMWL devices. The Microcon units were spun for 7 minutes at 12,000 x g. The concentrated DNA sample (retentate) was diluted

again with TE buffer (to 500 µL or 2 mL, depending on the device used) and spun once more, as described above. The dilution step was repeated a third time. After the third concentration spin, the retentate was collected by placing the sample reservoirs upside down in new vials and spinning the units for 1 minute at 1,000 x g. The concentrated DNA was run on a 0.9% SeaKem® GTG agarose gel (FMC) and stained with ethidium bromide to monitor sample integrity (Figure 1). DNA bands in lane 1 (starting material) are indistinguishable from the bands on lanes 2 and 3, which were spun repeatedly in the ultrafiltration device. There is no evidence that any supercoiled DNA was converted to the relaxed or linear forms during the concentration procedure, which would be the case had single-strand nicks been introduced during centrifugation.

Discrete Size Plasmids The next set of experiments monitored the effect of centrifugal ultrafiltration on single population, discrete size plasmids. The plasmids used were pBR322 (4,361 bp; New England Biolabs, Inc.), pSPT18 (3,104 bp; Boehringer Mannheim) and pXTl (10,400 bp; Stratagene). Samples (1 µg) of each plasmid were spun in Microcon 30K NMWL units, as described previously. The starting material and retentates were run on a 1% agarose gel and stained with ethidium bromide.

Figure 1. Supercoiled DNA Ladder.

Lane 1: Starting material Lanes 2, 3: Retentate from DNA ladder spun 3 times at 12,000 x g in Microcon 30K NMWL device

1

2

3

The results are shown in Figure 2. As in the case of the DNA Ladder, the concentrated samples (lanes 2, 4 and 6) appear to be identical with their corresponding starting material. Again, there is no evidence of conversion of the supercoiled form to the relaxed form after exposure to g forces of 5,000 x g for 90 minutes and 12,000 x g for 21 minutes during the concentration spins.

Genomic Size DNA To monitor the integrity of molecules in the size range of genomic DNA after centrifugation, lambda DNA (49 kb; Boehringer Mannheim, Indianapolis, IN) and Bsu36 l digested BacPAK6 DNA (125 kb) was used. The samples were diluted and centrifuged as described above. The retentates and starting material were run on a 1% agarose gel in a CHEF-DR® II pulsed field electrophoresis system Bio-Rad, Richmond, CA). Electrophoresis was performed at 200 V at 14 °C in 0.5X TBE with ramped pulse from 1 to 6 seconds over 14 hours. The results with the lambda DNA mimic those of the other samples run in these sets of experiments. No adverse effects are noted after spinning the DNA at g forces up to 12,000 x g in the ultrafiltration units. However, the larger BacPAK ®6 DNA does show some degradation after ultrafiltration at both 5,000 and 12,000 x g (Figure 3, smearing in lane 4). Although a large percentage of the sample appears to be intact, there was loss of integrity of the BacPAK6 DNA sample after the concentration procedure.

Figure 2.

1

2

3

4

5

6

Specific plasmid DNA

Lane 1: pBR322 starting material Lane 2: pBR322 spun in Microcon 30K NMWL device Lane 3: pSPT18 starting material Lane 4: pSPT18 spun in Microcon 30K NMWL device Lane 5: pXT1 starting material Lane 6: pXT1 spun in Microcon 30K NMWL device

Figure 3.

1

Large fragment DNA

Lane 1: Lambda DNA starting material Lane 2: Lambda DNA spun in Microcon 30K NMWL device Lane 3: BacPAK DNA starting material Lane 4: BacPAK DNA spun in Microcon 30K NMWL device

Conclusions

References

DNA samples of up to 49 kb were concentrated repeatedly without any loss of sample integrity. Some loss of integrity was observed with a 125 kb sample, although it was not complete and represents a small percentage of the total DNA in the sample. For large fragments of DNA, centrifugal ultrafiltration provides a fast and efficient method to concentrate or desalt the sample. It results in high recovery of intact product.

1. Takagi S, Kimura M, Katsuki M. BioTechniques 1993;14(2):218–21.

2

3

4

y t i r g e t n I A N D t n e m g a r F e g r a L n o n o i t a r t l i f a r t l U l a g u f i r t n e C f o t c e f f E •

2. PCR is covered by U.S. patents issued to Hoffmann-LaRoche, Inc. 3. Sheng N, Zhang J, Whitton JL, McKee T. BioTechniques 1993;14(5):781–4. 4. Ruano G, Pagliaro EM, Schwartz TR, Lamy K, Messina D, Gaensslen RE, Lee HC. BioTechniques 1992;13(2):266–74.


79

DNA Extraction from Agarose Gels with Montage Gel Extraction Kit or Ultrafree-DA Centrifugal Filters Introduction

s l e G e s o r a g A m o r f n o i t c a r t x E A N D

The Ultrafree-DA device is designed to recover 100 to 10,000 bp DNA from agarose gel slices in one 10-minute spin. It consists of a pre-assembled sample filter cup with an agarose gel nebulizer and a microcentrifuge vial. The device uses gel compression to extract DNA from the agarose. Centrifugal force collapses the gel structure, drives the agarose through a small orifice in the gel nebulizer and captures the resultant gel slurry in the sample filter cup. As the agarose is compressed at 5,000 x g, DNA is extruded from the gel’s pores. The gel matrix is retained by the microporous membrane, and the DNA passes freely through the membrane. DNA can then be recovered in the filtrate vial. The Montage Gel Extraction Kit consists of 50 Ultrafree-DA centrifugal filters as well as a modified TAE buffer that allows the casting and running of the gel from which the DNA fragment is to be extracted.

DNA prepared with the Ultrafree-DA centrifugal filter requires no further purification for most applications, including cloning and radioisotopic or fluorescent DNA sequencing. Since agarose gel electrophoresis has high resolving power, the small and large non-specific amplification products that frequently interfere with cloning and sequencing after PCR (polymerase chain reaction) are completely removed from the product.

Materials • Microcentrifuge • Pre-assembled Ultrafree-DA centrifugal filter device or Montage Gel Extraction Kit • Modified TAE* electrophoresis buffer (40 mM Tris-acetate, pH 8.0, 0.1 mM Na2EDTA) • SeaKem agarose (FMC BioProducts) or equivalent • Long-wavelength UV lamp • Scalpel or razor blade


*Modified TAE is recommended rather than TBE for the following reasons: (1) TBE buffer strongly inhibits DNA sequencing reactions while modified TAE buffer does not. (2) Modified TAE has 0.1 mM Na 2 EDTA while regular TAE has 1.0 mM Na2 EDTA. The EDTA level at 0.1 mM Na2 EDTA will not interfere with the magnesium concentration in sequencing reactions and other downstream enzymatic treatments, many of which are dependent on magnesium.

80

Procedure 1. Electrophorese 30 µL of PCR product or other DNA through a <1.25% ordinary agarose gel, prepared in modified TAE buffer with ethidium bromide (0.5 µg/mL). 2. Locate the band of interest with a long wavelength UV lamp or transilluminator. With a razor blade or scalpel, cut out the slice of agarose (<100 µL or 100 mg) containing the band of interest. Trim any away excess agarose from band. 3. Place the gel slice into the gel nebulizer/sample filter cup/filtrate vial assembly and seal the device with the cap attached to vial.

4. Spin at 5,000 x g for 10 minutes. Centrifugation forces the agarose through the gel nebulizer, converting it to a fine slurry that is captured by the sample filter cup. Extruded DNA in electrophoresis buffer passes through the microporous membrane in the sample filter cup and collects in the filtrate vial. 5. DNA in the filtrate is now ready for sequencing or cloning without further purification. Discard the gel nebulizer and sample filter cup and store the DNA in the capped filtrate vial.

Results Gel compression is a quick and easy technique for recovering DNA from an agarose gel slice.

Table 1. Effect of gel disruption on typical DNA recoveries from agarose gels

DNA Size (bp)

% DNA Recovered from Intact Gel

%DNA Recovered from Gel Disrupted by Gel Nebulizer

100 400

74 39

78 ND

700 1000 2027 4361 9416 23130

43 55 * 14 * *

71 77 47 35 32 29

* = Not detectable

s l e G e s o r a g A m o r f n o i t c a r t x E A N D • S D I C A C I E L C U N R O F S L O C O T O R P

81

PCR Purification with Montage PCR Filter Units Introduction After polymerase chain reaction (PCR)1, amplified DNA must be separated from excess reaction components that can interfere with subsequent manipulations such as cloning or sequencing. The Montage PCR filter unit is a single-use device that simplifies the purification of PCR products. It concentrates amplified DNA and removes primers and unincorporated dNTPs, while providing excellent capacity, high recovery, and high purity. The method is quick and highly reproducible, which makes it ideal for processing one-up or multiple samples in middle-throughput operations.

Materials • Variable speed microcentrifuge • Montage PCR filter unit • Purified water (such as Milli-Q water) or TE buffer • PCR reaction (aqueous phase) n o i t a c i f i r u P R C P • S D I C A C I E L C U N R O F S L O C O T O R P

Method 1. Insert the Montage PCR sample reservoir into one of the two vials provided. 2. Fill the reservoir with 300 µL purified water or TE buffer. Add 100 µL PCR reaction to the reservoir (step 1). Smaller volumes of PCR product may be used, but the volume should be adjusted to a final volume of 400 µL. 3. Spin the Montage PCR device at 1000 x g for 15 minutes (step 2). NOTE: For optimal recovery, do not centrifuge longer than the specified 15 minutes or greater than 1000 x g. 4. To recover DNA, remove the sample reservoir from filtrate collection vial and place in a clean vial. 5. Add 20 µL purified water or TE buffer to sample reservoir. 6. Invert the reservoir and spin at 1000 x g for 2 minutes to retrieve the purified PCR (step 3).

82

Figure 1. Primer DNA Salt dNTP

Step 1

Step 2

Step 3

Results Montage PCR filter units were used to purify 100 µL PCR reactions according to the specified protocol. After recovering the DNA fragments (n = 10) using a reverse spin, the samples were separated by agarose gel electrophoresis. Recoveries of the various PCR products were determined by densitometry (Figure 1). The Montage PCR filter unit is a convenient method for single-sample PCR purification. The high-performance device purifies PCR products in a single centrifugation step. Purified samples are ready for downstream applications with no additional purification steps.

References 1. PCR is covered by U.S. patents issued to Hoffmann-LaRoche, Inc.

Figure 2. 100% 80% y r e v o c e R t n e c r e P

60% 40% 20% 0% 137 bp

301 bp

500 bp

657 bp

1159 bp

Purification of PCR Products Using Montage PCR Filters

Figure 3.

n o i t a c i f i r u P R C P •

Typical electropherogram shows an 1159 bp PCR product purified with a Montage PCR filter unit. Note the uniform signal intensity and long read lengths. Purified samples are ready for cloning or sequencing with no additional purification steps.


83

Enzyme Removal with Micropure-EZ Centrifugal Filters Introduction The Micropure-EZ device is a convenient device for removing enzymes from double-stranded (ds) DNA (20 bp to >50,000 bp) solutions in a single centrifugation. It can be used to remove enzymes whenever heat inactivation or phenol/chloroform extraction is impossible or impractical. After an enzyme reaction, the modified DNA solution containing dilute protein is simply pipetted into a Micropure-EZ device that has been inserted into a vial (which is supplied) and centrifuged for 1 minute at 12,000–14,000 x g. The DNA passes freely into the vial (typically with 85–90% DNA recovery) while the enzyme remains bound to the proprietary membrane in the Micropure-EZ device. Removal is defined as the complete absence of detectable enzyme activity or, as in the isolated case of T4 polynucleotide kinase, inconsequential residual activity (<0.08%). For simultaneous concentration of the enzyme-free DNA, place the Micropure-EZ device into a Microcon microconcentrator l a v o m e R e m y z n E

Figure 1. DNA

Remove Enzyme

Enzyme

Salt

Micropure-EZ


Restriction and other enzymes are absorbed by Micropure-EZ. dsDNA passes freely.

Remove Enzyme

Micropure-EZ

Restriction and other enzymes are absorbed by Micropure-EZ. dsDNA passes freely.

Concentrate DNA

DNA is retained by ultrafilter in Microcon. Salts pass freely.

Microcon Assembly

Vial Recover

85% DNA

≥

30 seconds

Operation of Micropure-EZ device

3 minutes

(Figure 1) before centrifuging again. The purified DNA is suitable for cloning or for other enzymatic manipulation. While most enzymes were removed by the Micropure-EZ device (Table 1, on page 86), Millipore felt it was equally important to notify customers of several enzymes that were not removed (Table 2 , on page 86). Millipore scientists were very careful to rule out enzyme inhibition as a mechanism. The sensitivity of each assay to detect trace amounts of restriction enzymes in MicropureEZ filtrates was determined by carrying out a dilution series (this is the method used by restriction enzyme manufacturers to estimate total activity).

Method In order to ensure that each enzyme assay was detecting trace amounts of restriction enzymes in Micropure-EZ filtrates, serial dilutions of Bgl I (representing 2 units, 0.4 units, 0.08 units, and 0.016 units of total activity in 10 µL) were carried out in a reaction mix* prepared with either sterile DI water (sdH2O) or with sdH2O that had been filtered through Micropure-EZ devices and then incubated. Comparison of the resultant restriction digests after agarose gel electrophoreses verified that aqueous extractables from Micropure-EZ devices did not inhibit the enzyme. Also, these standard curves determined the sensitivity of the enzyme assay (i.e., the theoretical amount of residual enzyme activity that could be detected, if present). To stress test Micropure-EZ devices sufficiently with enzymes and DNA, several extreme operating conditions were used consistently. Micropure-EZ devices were challenged with 50 units of Bgl I in the presence of decoy DNA (1 µg of pBR322) and 5 µg of bovine serum albumin (BSA). Devices were spun at 14,000 x g for 30 seconds. The filtrates were then assayed for residual enzyme act ivity by adding 1 µL of pUC19 DNA and 0.5 µL of 100X BSA to the filtrate, mixing thoroughly and incubating

*15.8 μL of sdH 2 O, 2 μL of 10X NEBuffer 2, 0.2 μL of 100X BSA, and 2 μL of Bgl I (10 units/μL)

84

at 37 °C for 1 hour. After a brief centrifugation to collect the condensate at the bottom of the microcentrifuge tubes, 1 µL of 0.5 M disodium EDTA and 10 µL of 5X loading buffer were added to stop the reaction. The negative control was a portion of DNA master mix. Control digests of pBR322 DNA and pUC19 DNA were carried out separately.

Results and Discussion The Bgl I standard curve made with the MicropureEZ-filtered sdH2O was indistinguishable from the standard curve made with untreated sdH2O. The plasmid DNA (1 µg of pUC19 and 1 µg of pBR322) was cut to completion by 2 units of Bgl I after one hour at 37 °C (Figure 2, lanes 1 and 6). None of the enzymes shown in Tables 1 or 2 was measurably inhibited by Micropure-EZ-filtered sdH2O. The standard curves indicated that as little as 0.08 units of Bgl I could be detected by this assay (lanes 3 and 8). The devices removed the 50 units of Bgl I as evidenced by an absence of detectable activity in the filtrates. In the lanes corresponding to the Bgl I challenged Micropure-EZ inserts, some activity was observed against the decoy pBR322 DNA, as expected during its brief exposure to Bgl I (particularly evident in lane 14). However, the pUC19,

which was added directly to the filtrate and incubated, appeared completely intact (lanes 11–14). Since the standard curve indicated that 0.08 units would be detected if it were present, we calculated that at least 99.8% of Bgl I was removed and/or inactivated. Removal, rather than inactivation, is the probable mechanism by which Micropure-EZ devices operate, since in separate experiments we were unable to detect any enzyme (irrespective of activity) in Micropure-EZ filtrates using very sensitive HPLC methods (data not shown).

Summary Enzyme removal and excellent DNA recovery are accomplished with Micropure-EZ devices in a single 60-second spin without any pre-wetting, binding, washing or elution step. Cumbersome phenol/ chloroform extraction and the associated hazardous waste accumulation are avoided. An additional line of evidence for the suitability of Micropure-EZ devices in molecular biology applications is that restriction digest purified with Micropure-EZ devices alone are readily ligated and cloned into plasmid DNA (M.H.A.L., unpublished observations). Micropure-EZ devices used alone or with Microcon devices permit rapid and efficient sequential enzymatic DNA manipulations.

Figure 2. Analytical agarose gel, demonstrating the detection limits for Bgl I and Bgl I removal by Micropure-EZ devices.

Lanes 1–4:

Serial dilutions of BgI I representing 2 units, 0.4 units, 0.08 units, and 0.016 units of activity (respectively) against the DNA master mix prepared in non-filtered sdH 2O Undigested DNA master mix used as substrate for the standard curve containing pUC19 and pBR322 DNA Lane 5: Inhibition control: serial dilutions of BgI I representing 2 units, 0.,4 units, 0.08 units, and 0.016 units of activity (respectively) Lanes 6–9: against the DNA master mix prepared in Micropure-EZ-filtered sdH 2O Lane 10: Molecular weight standard: 1 kb DNA ladder (Gibco-BRL, Gaithersburg, MD) Lanes 11–14: Filtrates incubated with pUC19 DNA after challenging Micropure-EZ devices with 50 units of BgI I, BSA and decoy pBR322 DNA Uncut pBR322 Lane 15: Lane 16: BgI I–cut pBR322 Uncut pUC19 Lane 17: BgI I–cut pUC19 Lane 18:

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

l a v o m e R e m y z n E • S D I C A C I E L C U N R O F S L O C O T O R P

18

85

Table 1. Enzymes removed by Micropure-EZ devices Enzyme

Challenge

Enzyme

Challenge

Enzyme

AMV reverse transcriptase

50 U

Bgl I

50 U

Nru I

50 U

Calf intestinal alkaline phosphatase

10 U

Bsi W I BssH II

70 U 20 U

Pst I

100 U

Sac I

100 U

DNase I (bovine pancreas) 10 U Exonuclease III (E. coli ) 100 U

Bst N I

50 U

Dpn I

100 U

Sac II Sal I

100 U 100 U

MMLV reverse transcriptase

Eco R I

100 U

Hae III

100 U

Sca I Sph I

50 U 25 U

Hinc II Hind III

50 U 100 U

Sst I

50 U

Xho I

100 U

Hpa I

25 U

Kpn I

50 U

Mbo I

25 U

Mlu I Nco I

50 U 50 U

Nde I

100 U

600 U

Mung bean nuclease

50 U

Proteinase K (Amresco)

5 µg

T4 DNA ligase T4 DNA polymerase

2,000 U* 15 U

T4 polynucleotide kinase** 50 U Taq DNA polymerase 5U Terminal deoxynucleotidyl transferase

45 U

Acc I

50 U

Apa I

100 U

BamH I

100 U

Bcl I

50 U

Ngo M I Nhe I

50 U 25 U

Not I

50 U

In tests, all enzymes were removed from solutions containing DNA or RNA (in the case of RNase A). Five µg of bovine serum albumin were also present during removal of restriction enzymes. Removal was indicated by undetectable or inconsequential enzyme activity in filtrate (<0.08% residual in the case of T4 polynucleotide kinase).

*New England Biolabs unit definition **T4 polynucelotide kinase is not recommended with oligonucleotides (dsDNA only). The results suggest that this kinase mediates the binding of oligos to the membrane in Micropure-EZ device, causing oligo loss.

Table 2. Enzymes not removed by Micropure-EZ devices l a v o m e R e m y z n E • S D I s C d A i c C A I E c i L e C l c U u N N R O • F S L O C O T O R P

Enzyme

Challenge

Enzyme

Challenge

Bacterial alkaline phosphatase

0.6 U

Eae I Eco R V

15 U 50 U

DNA polymerase I (Klenow)

20 U

Hinf I

50 U

Exonuclease I

50 U

Pfu DNA polymerase

2.5 U

Msp I Pvu I

100 U 25 U

Vent™ DNA polymerase Shrimp alkaline phosphatase RNase A (bovine pancreas) ApaL I Bgl II

10 U 50 U

Bso B I

50 U

Cla I

25 U

4U 1U 1 µg

Pvu II Sau 3A I

50 U 20 U

Sfi I

50 U

Sma I

25 U

Xba I

50 U

In tests, Micropure-EZ devices did not remove the indicated number of units of the listed enzymes. It may be effective in removing a lower number of units.

*T4 RNA ligase is not recommended. Results suggest this ligase mediates binding of nucleic acids to the membrane in Micropure-EZ devices, causing sample loss.

86

Challenge

Protocols for Virus Concentration

Concentration of Bacteriophage Using Ultrafiltration . . . . . . . . . . . . . . . . . . . . 88 Concentration of Animal Viruses Using Ultrafiltration . . . . . . . . . . . . . . . . . . . . 91

N O I T A R T N E C N O C S U R I V R O F S L O C O T O R P

87

Concentration of Bacteriophage Using Ultrafiltration Introduction Bacteriophages were first discovered nearly a century ago1. Since then, phage biology has drawn researchers’ attention because of their potent bactericidal capacity that can be used to treat human infections 2. Bacteriophage and animal n o i t a r t l i f a r t l U g n i s U e g a h p o i r e t c a B f o n o i t a r t n e c n o C • N O I T A R T N E C N O C S U R I V R O F S L O C O T O R P

88

viruses have been successfully utilized as DNA carriers, or cloning vectors. The advantage to lambda-based vectors is that they can carry fragments of DNA up to 25,000 bp. Another application, phage display, is a novel genetic selection system for cloning proteins from cDNA libraries. In addition, there are many similarities between bacteriophages and animal cell viruses. Thus, bacteriophages can be viewed as model systems for animal cell viruses. The standard bacteriophage propagation protocol normally involves infection of the host bacterial strain, 12–16 hours of bacterial growth, followed by the phage recovery from the supernatant. Purification of phage usually includes a

density gradient centrifugation, followed by extensive dialysis3,4. Phage titers are then determined by plaque assays. For some experiments, high titer bacteriophage stock is desired. Traditionally, phage particles were concentrated by polyeteylene glycol (PEG) and dextran sulfate precipitation, precipitation by acid, or differential or equilibrium centrifugation5. Recently, different modifications of the Sambrook method are being used6. Phage is concentrated by precipitation with sodium chloride and PEG and resuspended to the desired titer . The process requires 6–12

hours of incubation with PEG and a lengthy pellet resuspension process to achieve high particle recovery 7,8.

Ultrafiltration is an alternative method to achieve high bacteriophage titer. When complete purification from cell and media proteins is not required, phage can be quickly concentrated using centrifugal ultrafiltration9. Ultrafiltration can also be used to concentrate phage from environmental sources10. For larger volume lysates, recirculating tangential flow filtration (TFF) is a better choice11,12. Correct membrane material, pore size, centrifugation speed and final retentate volume are critical to obtaining high phage recovery and maintaining its infectivity. This protocol describes the use of Millipore ultrafiltration devices to concentrate Pseudomonas pseudoalcaligenes bacteriophage phi-6, an enveloped dsRNA virus. The virions are spherical and 86 nm in diameter.

Method Phi-6 production Note: All manipulations should be performed using aseptic technique in a laminar flow hood. 1. Grow fresh culture of Pseudomonas pseudoalcaligenes until it reaches the desired optical density of 0.1 absorbance units at 600 nm. 2. Inoculate the culture with enough phi-6 to achieve a multiplicity of infection (MOI) of 0.5. 3. Return the phi-6 containing Pseudomonas pseudoalcaligenes culture to the platform shaker and incubate at 26 °C, 125 RPM, for 18–24 hours. 4. After the overnight incubation, aliquot the culture into four 250 mL centrifuge bottles. 5. Centrifuge the bottles for 30 minutes at 16,000 x g. 6. Remove the supernatant and transfer to a sterile container.

Concentration using Amicon Ultra-4 or Ultra-15 devices with 50 kDa NMWL membrane 1. Clarify phi-6 supernatant using a Stericup filter unit (Millipore cat. no. SCGP U05 RE), a Steriflip filter unit (Millipore cat. no. SCGP 005 25), or a Millex-GP syringe filter (Millipore cat. no. SLGP 033 RB) with lowbinding 0.22 µm PES membrane. 2. Load appropriate volume of clarified phi-6 supernatant (4 or 15 mL) into an Amicon Ultra-4 device (Millipore cat. no. UFC8 050 24) or an Amicon Ultra-15 device (Millipore cat. no. UFC9 050 24) containing 50,000 NMWL regenerated cellulose membrane. 3. Centrifuge the device (4 or 15 mL) for 15 minutes at 1500 × g. 4. The recoverable volume should be 80–100 µL for Amicon Ultra-4 devices and 350–500 µL for Amicon Ultra-15 devices. Note: Avoid concentrating samples below recommended volumes as this may lead to decreased phage recovery.

Concentration using Pellicon XL50 device with PLCTK membrane and Millipore’s Labscale TFF system Note: The Labscale TFF system (Millipore cat. no. XX42 LSS 13) is not required to use Pellicon XL50. A peristaltic pump may alternatively be used (see Millipore user guide P60085). 1. Perform flush, integrity, and permeability tests on Pellicon XL50 device with PLCTK membrane (Millipore cat. no. PXCO 30C 50) as indicated in the Pellicon XL50 User Guide. 2. Remove reservoir cover and load up to 500 mL phi-6 supernatant into reservoir of Labscale TFF system. 3. Ensure vent port is open by removing plug from VENT port and leaving open or installing a Millex filter. 4. Open tank outlet valve. 5. Turn on pump and increase speed until feed pressure reaches 20 psi.

6. Set retentate pressure by slowly adjusting retentate valve clockwise until retentate pressure is 10 psi. 7. Adjust pump speed and retentate valve restriction to achieve desired feed pressure of 30 psi and retentate pressure of 10 psi. Note: Do not exceed feed pressure of 60 psi. 8. Filter the solution until the desired concentration or volume is obtained. 9. Stop pump. 10. Disconnect pump outlet tubing from pump outlet port and place in product recovery collection vessel. 11. Disconnect retentate tubing from retentate in port. Fluid should now drain by gravity. If additional drainage is required, a syringe can be placed on the end of the retentate tube and fluid can be blown down. 12. Replace retentate tubing in retentate port. Reconnect pump outlet tubing. 13. Disconnect FEED IN tubing and place in collection vessel. Open tank outlet valve, turn pump speed up to drain reservoir. 14. Reconnect the pump outlet tubing to the FEED IN port.

Results Millipore ultrafiltration devices with Ultracel regenerated cellulose membrane offer low non-specific binding and high retentate recovery. The vertical membrane reduces concentration polarization for ultra-fast concentration times and is especially beneficial for turbid and viscous solutions. As shown in the protocol above, phage titer can be increased up to 30-fold in 15 minutes by centrifugal ultrafiltration for under 15 mL volumes. Amicon Ultra-4 and Ultra-15 devices with 50 kDa NMWL membranes can be used successfully to concentrate the phage phi-6 out of bacterial culture supernatant. The same devices with 100 kDa NMWL membranes are not recommended because they lead to a low virus recovery and allow virus to break through into the filtrate.

n o i t a r t l i f a r t l U g n i s U e g a h p o i r e t c a B f o n o i t a r t n e c n o C • N O I T A R T N E C N O C S U R I V R O F S L O C O T O R P

Note: Check system connections for leaks. Tighten connections as required.

89

For large volume concentration, tangential flow filtration (TFF) should be used. Table 1 shows phage concentration results using a Pellicon XL50 cassette with the Labscale TFF system. The Pellicon XL50 cassette couples Millipore’s ultrafiltration membranes with the linearly scalable TFF casset te for processing 50–1000 mL volumes. The Ultracel membrane with 30 kDa NMWL is recommended. Twenty-fold concentration of phage solution is usually achieved in 30–50 minutes.

5. Bachrach U, Friedmann A. Appl Microbiol 1971;22(4): 706-715.

References

9. Meile L, Abendschein P, Leisinger T. J Bacteriol 1990;172(6):3507-3508.

1. Pennazio S. Riv Biol 2006;99(1):103-29. n o i t a r t l i f a r t l U g n i s U e g a h p o i r e t c a B f o n o i t a r t n e c n o C • N O I T A R T N E C N O C S U R I V R O F S L O C O T O R P

90

6. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989 7. Bahns JT, Liu CM, Chen L. Protein Science 2004;13:2578-2587. 8. Gill JJ, et al. Chemother 2006;50(9): 2912-2918.

10. Middelboe M, et al. Aquat Microb Ecol 2003;33:1-10.

2. Fischetti VA, Nelson D, Schuch R. Nature Biotechnology 2006;24:1508-1511.

11. Rembhotkar GW, Khatri GS. Anal Biochem 1989;176(2):373-4.

3. Hansen MR, Mueller L, Pardi A. Nature Structural Biology 1998;5:1065-1074.

12. Alonso MC, Rodriguez J, Borrego J. J Internatl Microbiol 1999;2:227-232.

4. Zhilenkov EL, et al. Virology Journal 2006;3:50-55.

Table 1. Bacteriophage phi-6 concentration using Millipore ultrafiltration devices Amicon Ultra-4, 100 kDa

Amicon Ultra-4, 50 kDa

Amicon Ultra-15, 50 kDa

Pellicon XL50 TFF, 30 kDa

Initial volume (mL) Initial viral titer, pFU/mL

4 2.3 x 1010

4 2 x 1010

15 2.02 x 1010

500 1.9 x 107

Final volume (mL) Final viral titer, pFU/mL Concentration factor

0.1 2.06 x 1011 40

0.5 3.7 x 1011 8

0.5 7.8 x 1011 30

25 5.6 x 108 20

22 2.3 x 105

>100 <100

>100 <100

>100 7 x 103

Phage recovery (%)* Phage in the filtrate, pFU/mL

*We consistently observe higher than 100% recovery after phage concentration. This can be explained by increased infectivity of phage particles at higher concentrations and agrees with other published data12 .

Concentration of Animal Viruses Using Ultrafiltration Introduction Despite the rapid progress in genomics, the biological function of the majority of the human genes is still unknown. The identification of gene function is an increasingly relevant issue, especially in the search for new targets for improved human disease therapy. Functional genomics approaches, which aim at the identification of genes via phenotypes induced in biological systems, require measurement of gene function on the genomic scale in cell-based assays. cDNA expression libraries representing the population of expressed genes in a given cell/tissue type have classically been cloned into plasmids, which can be introduced into cells by transient transfection. However, transfection efficiency varies widely between cell types. Therefore, a variety of virus-derived vector systems have been developed for improved cDNA transduction, expression, and screening in mammalian cells. These systems include simian virus 40 replicons in COS cells1,2 and cDNA cloning and expression vectors derived from retrovirus3,4 baculovirus5, alphavirus6, human immunodeficiency virus7, vaccinia virus8, adenovirus9

Figure 1. Primary culture of rat cortical neurons infected with lentivirus

and adeno-associated virus (AAV)10. Challenges associated with the use of viral vectors in clinical trials include production of sufficient quantities of clinical grade material and maintaining biosafety of the viral vector. Production and purification methods for viral vectors vary both in terms of methodology and virus yield and quality. Generation and purification of viral vectors can be labor intensive, costly and may require special equipment (such as ultracentrifuges). The standard method for purification of adenovirus, lentivirus and AAV uses density gradient ultracentrifugation followed by extensive dialysis. More recently, column or membrane chromatographies have become the methods of choice for adenovirus purification11,12. The yield of virus normally is determined by plaque assay, TCID50 assay and more recently by ELISA assay. For most applications, recombinant virus must be purified to a high titer. In order to achieve high titer stock, it is usually necessary to concentrate purified virus particles. This Protocol describes the use of Millipore centrifugal ultrafiltration devices for concentration of three most common virus types: lentivirus, adenovirus and adeno-associated virus (AAV).

•

Method 1. Packaging cells: Host cells are plated to achieve 80–85% confluence 2. Transfection: Cells are either transfected with recombinant plasmid(s), or infected with viable virus particles.

For virus propagation, HEK-293T cells were transfected with helper plasmids and lentiviral plasmid expressing copGFP (System Bioscience). 48 hours after transfection viral supernatant was collected and 10 mL was concentrated using Amicon Ultra-15 100 kDa MWCO membrane to 200 µL. 50 µL of concentrated viral stock was used for infection of 10E6 cells. Data courtesy of Drs. Leila Aminova and Ambreena Siddiq, Neuroprotection Laboratory of Beth Israel Medical Center.

n o i t a r t l i f a r t l U g n i s U s e s u r i V l a m i n A f o n o i t a r t n e c n o C

3. Harvesting virus: Depending on the virus, either cell supernatant or pelleted cells, or both, are used to purify the recombinant virions. Three to four rounds of freeze/thaw cycles are usually applied to release virus from cells. Cell debris is then separated from cell lysate by centrifugation.

N O I T A R T N E C N O C S U R I V R O F S L O C O T O R P

4. Virus purification: Virus is purified from cell lysate by density gradient ultracentrifugation or column/membrane chromatography. In some cases, affinity chromatography with heparin or mucin sepharose13,14,15 can be used. 91

Purified virus solutions obtained via these methods are then buffer exchanged using either passive dialysis or diafiltration into an appropriate storage solution.

Virus Concentration

n o i t a r t l i f a r t l U g n i s U s e s u r i V l a m i n A f o n o i t a r t n e c n o C • N O I T A R T N E C N O C S U R I V R O F S L O C O T O R P

92

In order to achieve a high titer of viral stock, the purified virus can be concentrated using centrifugal ultrafiltration. The correct choice of device, membrane material, MWCO, centrifugation speed, centrifugation time and buffer composition are critical for high recovery of infective viral particles. Amicon Ultra centrifugal ultrafiltration devices with a 50 KDa NMWL Ultracel regenerated cellulose membrane have been demonstrated to successfully concentrate adenovirus and AAV solutions. For lentivirus concentration, devices with both 50 and 100 kDa membranes can be used.

Table 1 shows typical results of concentration of adenovirus, lentivirus and AAV from both crude cell lysate and virus purified by chromatography. It is important to remember that most of the viruses cannot be efficiently concentrated and stored in low salt buffers. Centrifugal concentration of the virus in <200 mM salt solutions will lead to virus aggregation and decreased infectivity. An example of a recommended storage solution is 20 mM Tris, pH 8.0, 250 mM NaCl, 10 mM MgCl2, 5% sorbitol and 0.001% PF68 (pluronic acid)16. Concentrated viral stock can also be filter sterilized through 0.22 µm filters. For example, Steriflip®-GP filter units (cat. no. SCGP 005 25) can be used for volumes of 50 mL or less or Ultrafree-MC GV (cat. no. UFC3 0GV 0S) can be used for volumes of <0.5 mL.

Table 1. Concentration of virus using Millipore ultrafiltration devices Starting Virus Titer

Starting Volume (mL)

Final volume (µL)

Spin Time at 1500 x g (min)

Recovery (%)*

Concentration Factor

Starting solution

Device

Adenovirus in crude cell lysate Adenovirus purified, in 1M salt buffer

Amicon Ultra-4, 50 kDa MW Amicon Ultra-4, 50 kDa MW

1 x 108

4

150

20

>100

27X

1 x 108

4

80

10

85

45X

AAV in crude cell lysate AAV in crude cell lysate, diluted in 100 mM Tris, 200 mM NaCl buffer

Amicon Ultra-4, 50 kDa MW Amicon Ultra-15, 50 kDa MW

2 x 107

4

200

35

50

20X

2 x 107

10

400

45

76

25X

AAV in crude cell lysate, diluted in 100 mM Tris, 200 mM NaCl buffer Lentivirus in cell culture supernatant

Amicon Ultra-4, 50 kDa MW

0.5 x 107

2

200

20

100

20X

Amicon Ultra-4, 100 kDa MW

9 x 104

4

40

30

>100

100X

Lentivirus in cell culture supernatant Lentivirus in cell culture supernatant

Amicon Ultra-4, 50 kDa MW Centricon 70 Plus, 100 KDa MW

9 x 104

4

40

30

93

100X

8 x 103

65

800

30

81

80X

*We do not recommend concentrating the v irus to higher than 10 13 particles/mL due to potential virus aggregation 17 .

References 1. Gearing DP, et al. EMBO J 1989;8:3667. 2. Yokota T, et al. Proc Natl Acad Sci 1984;81: 1070. 3. Kitamura T, et al. Proc Natl Acad Sci 1995; 92:9146. 4. Shaughnessy L, et al. J Mol Neurosci 2004; 24:23. 5. Granziero L, et al. J Immunol Methods 1997; 203:131. 6. Koller D, et al. Nat Biotechnol 2001;19:851. 7. Van Maanen M, et al. Mol Ther 2003;8:167. 8. Smith ES, et al. Methods Mol Biol 2004; 269:65. 9. Elahi SM, et al. Gene Ther 2002;9:1238. 10. Choi VW, et al. Curr Gene Ther 2005; 5:299. 11. Blanche F, et al. Gene Therapy 2000;7: 1055-1062. 12. Shabram PW, et al. Hum Gene Therapy 1997; 9:453-465. 13. Clark KR, et al. Hum Gene Therapy 1999; 10:1031-1039. 14. Zolotukhin S, et al. Gene Therapy 1999;6: 973-985. 15. Auricchio A, et al. Hum Gene Therapy 2001; 12:71-76. 16. Alejandra E, Arbetman, AE, et al. Journal of Virology 2005;79(24):15238-15245. 17. Galdiero F. Arch Virol. 1979;59(1-2):99-105.

n o i t a r t l i f a r t l U g n i s U s e s u r i V l a m i n A f o n o i t a r t n e c n o C • N O I T A R T N E C N O C S U R I V R O F S L O C O T O R P

93

High Throughput Applications The MultiScreen filter plate with Ultracel-10 membrane provides a new method for high throughput sample prep. The ultrafiltration-based filter plate is designed for use with centrifugation and is compatible with standard microtiter plates, instrumentation, and liquid handling equipment. All of the publications summarized below can be supplied by your local Millipore office or downloaded from www.millipore.com/ultracel.

Nucleic Acid Purification and Concentration AN1040EN00 The MultiScreen Filter Plate with Ultracel-10 membrane can be used to purify and concentrate single-stranded oligonucleotides and doublestranded DNA fragments, as well as plasmid and genomic DNA.

Concentration of Proteins in Cell Lysate AN1424EN00 The MultiScreen filter plate with Ultracel-10 membrane can be used to concentrate whole cell lysates without loss of protein and with high reproducibility across the plate. Applications include parallel protein purification, protein concentration and buffer exchange in cell lysates for subsequent separation or assaying. s n o i t a c i l p p A t u p h g u o r h T h g i H • X I D N E P P A

94

MultiScreen Filter Plate with Ultracel-10 ultrafiltration membrane

Purification of Serum Peptides for Biomarkers Research AN2010EN00 This study is a high throughput version of the application note on page 42 of the Protocols section of this handbook.

Enzymatic Activity Recovery AN2011EN00 This study demonstrates the use of the MultiScreen plate with Ultracel-10 membrane for concentrating alkaline phosphatase without loss of biological activity and with high reproducibility across the plate.

Glossary Asymmetric Membrane 1 Membrane constituted of two or more structural planes of non-identical morphologies. Batch Process A fixed volume of solution contained in a tank to which the concentrate is returned during the process. Composite Membrane 1 Membrane having chemically or structurally distinct layers. Concentration Polarization Accumulation of rejected solute on the membrane surface. Depends on interactions of pressure, viscosity, crossflow (tangential) velocity, fluid flow conditions, flow channel conditions and temperatures. Crossflow (Tangential Flow) Solution flows across (tangential to) a membrane surface. Facilitates back diffusion of solute from that surface into the bulk solution, counteracting concentration polarization. Diafiltration Removal of small components from the retained species during ultrafiltration by adding water or buffer solution to the retentate. See page 7 for further discussion. Dialysis Diffusive transport of ions or other small molecules through a membrane barrier that contacts solvent on both sides. Downstream1 Side of a membrane from which the permeate emerges. Feed (Sample) The starting solution (sometimes the solution remaining upstream of the membrane). Fluid Velocity The flow rate of solution across the membrane surface in cross (tangential) flow. Related to hydraulic pressure drop. Flux The filtration rate through the membrane per unit area.

Fouling Irreversible decline in membrane flux due to deposition and accumulation of submicron particles and solutes on the membrane surface. Also, crystallization and precipitation of small solutes on the surface and in the pores of the membrane. Not to be confused with concentration polarization. Membrane 1 Structure having lateral dimensions much greater than its thickness, through which mass transfer may occur under a variety of driving forces. Molecular Weight Cut-off (MWCO) See Nominal Molecular Weight Limit. Nanofiltration1 Pressure-driven membrane-based separation process in which particles and dissolved molecules smaller than about 2 nm are rejected. Nucleotide Cut-off (NCO) The number of nucleotides in a DNA fragment (single- or double-stranded) at which 90% of the fragment is retained by the membrane. Nominal Molecular Weight Limit (NMWL) The molecular weight at which at least 90% of a globular solute of that MW is retained by the membrane. Permeate (Filtrate, Ultrafiltrate) The solution passing through the membrane, containing solvent and solutes not retained by the membrane. Plugging Accumulation of debris on the fluid flow path, restricting or blocking flow. Rejection The fraction of solute held back by the membrane. Can be measured at any point in the process or averaged over the run. Retentate (Reject Stream, Concentrate) The solution containing the retained (rejected) species. Retention Factor 1 (rF) Parameter defined as one minus the ratio of permeate concentration to the retentate concentration of a component. Note: rF = 1 – [p]/[r] where [p] = concentration of solute in permeate, and [r] = concentration of solute in retentate.

Reverse Osmosis1 Liquid-phase pressure-driven separation process in which applied transmembrane pressure causes selective movement of solvent against its osmotic pressure difference. Tangential Flow Filtration (TFF) Flow through a membrane device in which the fluid on the upstream side moves parallel to the membrane surface. Transmembrane Pressure (TMP) The driving force in ultrafiltration. In a stirred cell, equivalent to gas pressure. In centrifugal devices, it is related to g-force. In a flowing system, TMP decreases as the stream moves from inlet to outlet. Average TMP = [(Pin + Pout)/2] – P permeate Ultrafiltration1 Pressure-driven membrane-based separation process in which particles and dissolved molecules smaller than 0.1 µm and larger than about 2 nm are rejected. Yield Amount of species recovered at the end of the process as a percentage of the amount present in the feed solution.

References 1. Terminology for membranes and membrane processes (IUPAC Recommendations 1996). IUPAC, Journal of Membrane Science.1996;120(1): 149–59.

y r a s s o l G • X I D N E P P A

95

Acknowledgements Millipore also wishes to acknowledge the following individuals for their contributions: Eduardo Vottero University of British Columbia Peter A. Lemaire and Dr. James Cole University of Connecticut Gary Smejkal Proteome Systems Woburn, MA Leonid Kryazhev Genome Quebec Montreal, Canada Mathew L. Thakur Thomas Jefferson University Hospital Philadelphia, PA Mark Merchant Helena Laboratories Beaumont, TX X I D N E P P A

Department of Forensic Biology New York City Patrick O. Brown, Max Diehn, Ash Alizadeh Stanford University School of Medicine Dr. Sophia N. Karagiannis GKT School of Biomedical Sciences London

96

Patent PCR is covered by US patents issued to Hoffmann-LaRoche, Inc.

Trademarks Millipore, Amicon, Biomax, Centricon, Centriprep, Durapore, Immobilon, Labscale, Microcon, Micropure, Milli-Q, Minicon, Montage, MultiScreen, Pellicon, Prep/Scale, PROSEP, Steriflip, Ultracel, Ultrafree, Zip, and ZipTip are trademarks of Millipore Corporation. AmpFLSTR, BigDye and Voyager-DE are trademarks of Applera Corporation or a subsidiary. BacPAK and Talon are trademarks of Becton, Dickinson and Company. Biofuge is a trademark of Kendro Laboratory Products, Gmbh. CHEF-DR and Chelex are trademarks of Bio-Rad Laboratories. DryStrips, Sephacryl, Sepharose, and Storm are trademarks of GE Healthcare. GenePrint, Flexi and Riboprobe are trademarks of Promega Corporation. Kodak is a trademark of Eastman Kodak Company. MEGAscript is a trademark of Ambion Inc. NuPage, SimplyBlue, Superscript and Xcell Surelock are trademarks of Invitrogen Corporation. ProteoPrep is a trademark of Sigma-Aldrich Biotechnology LP. ProteomIQ, ElectrophoretIQ, IsoelectrIQ, and GelChips are trademarks of Proteome Systems. SeaKem is a trademark of FMC. SpectraFLUOR is a trademark of Tecan Group AG. SpectraMax is a trademark of Molecular Devices Corporation. Speed Vac is a trademark of Thermo Savant. SureBlue is a trademark of Kirkegaard & Perry Laboratories, Inc. SYBR is a trademark of Molecular Probes, Inc. Triton is a trademark of Union Carbide Chemicals & Plastics Technology Corporation. Tween is a trademark of Atlas Powder Company. Vent is a trademark of New England Biolabs, Inc.

81898709-Millipore-UF-Catalog.pdf

Recommend Documents