PROTEIN ASSAY USING THE BRADFORD METHOD Gison, Ashiea Lyka, Go, Jeanne Anne, Gonzales, Charisse Nicole, Grageda, Nicholle Anne, Lee, Aaron Group 5 2E Medical Technology Biochemistry Laboratory
ABSTRACT Quantitative determination is instrumental in many fields of protein study. One method for determination of protein concentration is the Bradford protein assay. It is a spectroscopic analytical procedure used to measure protein concentration in a solution. This procedure relies on the binding of Coomassie Brilliant Blue G250 dye to protein, in which protein concentration is proportionate to the dye. In this experiment, blank and 7 other test tubes were assigned with a certain volume of Bovine albumin standard and a volume of distilled water. The last test tube will constitute the unknown protein. Absorbance was measured using the Spectronic Genesys 5 Ultraviolet-visible spectrophotometer and protein concentration was computed using the dilution equation. The protein concentration of the unknown was determined by using two methods: linear regression method and graphical method
INTRODUCTION A rapid and accurate method for the estimation of protein concentration is essential in many fields of protein study. Quantitative determinations of proteins can be titrimetricelemental, gravimetric or spectroscopic. The Bradford assay has become the preferred method for quantifying protein in many laboratories. This spectroscopic technique is simpler, faster, and more sensitive than the Lowry method. Moreover, when compared with the Lowry method, it is subject to less interference by common reagents and non-protein components of biological samples. The Bradford assay relies on the binding of the dye Coomassie Brilliant Blue G250 to protein, in which the dye is proportional to the protein concentration. Without the protein, the solution is red-brown in its acidic solution and when protein binds, the pKa of the dye shifts causing it to turn blue. The assay is sensitive to about 20 to 200 μg protein. Thus, the quantity of protein can be estimated by determining the amount of dye in the blue ionic form. This is done by measuring the absorbance of the solution at 595 nm.
EXPERIMENTAL A. Compounds Tested (samples used): Bradford reagent, Bovine serum albumin (BSA) standard (100 µg/ml), unknown protein sample B. Procedure: In this experiment, 100 µg/ml concentration Bovine Serum Albumin (BSA) was used as the standard protein. To determine the protein concentration of an unknown sample, 7 dilutions were done to
make sure that that the protein concentration was within the range of the assay. Also, a “blank” or the test tube without the standard protein and an unknown protein was used. 1.5 mL of Bradford reagent which is composed of Coomasie Brilliant Blue G-250 dye, 95% ethanol and 85% phosphoric acid, was added in each test tube. The solution was mixed well by inversion or gentle vortex-mixing and left to stand for 5 minutes. The Spectronic Genesys 5 UV-VIS Spectrometer was used to measure absorbance. Each solution was placed in separate cuvettes. The absorbance results of the samples and standards against the reagent blank were measured within 1 hour after mixing. The albumin standard curve was constructed by plotting A595 against protein concentration and the concentration of the unknown was determined graphical method or by linear regression analysis.
RESULTS AND DISCUSSION The Bradford method is a spectroscopic analytical procedure used to measure protein concentration n a certain solution. The Ultraviolet-Visible spectrophotometer is then used to measure the absorbance of the standard and unknown protein. The spectrophotometer uses light in the visible and adjacent near ultraviolet and near-infrared ranges as shown in Figure 1 to produce it’s readings. The absorption in the visible range directly affects the perceived color of the chemicals involved.
obtain a more accurate result, the linear regression method was used to determine the protein concentration. The following solutions were used:
Figure1: wavelength-selectable, single-beam spectrophotometer schematic illustration
UV-Vis
After conducting these tests, we were able to come up with the following results as shown in the table below. Table I: Test results Conc. of BSA Tube No. (µg/ mL) A595 Blank
0
0
2
3.3
0.987
3
5.0
1.014
4
6.7
inconsistent*
5 6
8.3 10
1.155 1.186
7
12
1.202
8
13.3
0.501
*Results produced were inconsistently low, so the result was removed as per recommendation of the attending professor.
The concentration of the unknown protein was determined by two ways: first, by graphing the computed amount of protein concentration against absorbance and by using linear regression method. Through the graphical method the concentration of the unknown protein was determined, the protein concentration on the x-axis while absorbance was in the y-axis.
Graph 1: Test Results
Figure 2: Computation for Protein Concentration using Linear Equation
REFERENCES 1. Bradford, M. M. (2000). Analyt. Biochem. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. 72,248–254. 2. Bradford Method: Colorimetric Protein Assay Retrievedofromphttp://bioteachnology.co m/protein/bradford-method-colorimetricprotein-assay 3. Crisostomo, A. et. al(2010). Laboratory Manual in General Biochemistry. Isolation and Characterization of Proteins. South Triangle, Quezon City: C&E Publishing, Inc. Ultraviolet-visible spectrophotometry. Retrieved fromohttp://en.wikipedia.org/wiki/Ultraviole t%E2%80%93visible_spectroscopy 4. Noble, J.E.; Bailey, M.J.A. (2009). Methods Enzymol. Quantitation of Protein. Retrieved from http://en.wikipedia.org/wiki/Bradford_prot ein_assay 5. Zuo, S.-S. and Lundahl, P. (2000). Analyt. Biochem. A micro-Bradford membrane protein assay. 284, 162–164.
1.5 1
6. http://www.scribd.com/doc/49138231/Bra dford-Protein-Concentration-AssayFormal-Report
0.5 0 0
5
10
15
Due to some errors in the experiment, the most ideal line was not produced. The unknown protein concentration was determined by tracing the point in which the absorbance, 0.501, met the best-fit line of the graph. But the concentration result could only be approximated to around 13. lllIn order to
