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Experiment 7: Electrophoresis of Protei
Alex Foo (Partner: Matthew Crerar) 5673657 –
[email protected]
Bchm 317 – Group 3 TA: Da Duan November 10, 2009
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Introduction
Electrophoresis Electrophoresis is one of the most versatile tools in analytical biochemistry, a allows separation of proteins and other molecules based on a number of different
properties. However, in order to use electrophoresis effectively in the laboratory, on have a thorough understanding understanding of the mechanism of separation for different electrophoresis electrophoresis methods, and the strengths and limitations of associated with each
method. In this study, Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
PAGE) is used to separate proteins from samples of purified invertase, invertase, and to determ
the molecular mass of rat Lactate Dehydrogenase (LDH) isozymes. Cellulose Acetat
Electrophoresis Electrophoresis (CAE) is then used to resolve the different LDH isozymes. The prope and limitations of both techniques will be explored during the course of the study.
Material Mater ials s and Methods Metho ds:
All materials and methods used in this laboratory experiment are found in the Quee University 317 lab manual and were used without modification. Master your Bchm semester with [1], Scribd Read Free Foron 30this Days Sign up to vote title & The New York Times Useful Not useful Special offer for students: Only $4.99/month.
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Results:
*Note: all tables and figures are sh
the appendix
In this experiment, SDS-PAGE gel electrophoresis was used to determine the
molecular weight of Lactate Dehydrogenase Isozymes (LDH) from rat heart and mus mu
tissue. The molecular weight of carboxypeptidase carboxypeptidase was also measured. Molecular we
measurements were calculated calculated by comparing the distance each of the sample band
migrated to a standard lane loaded with peptides of known molecular weights, weights, as sh
figure 1. The 4 lysosome purification purification fractions from previous experiments were also
the same gel to qualitatively observe the purity of the various fractions. This is done
both observing the size and intensity of the invertase protein band, and the number intensity of other contaminating protein bands on the gel.
The distance migrate and mass of each of the standard proteins was tabulate
table 1, and the data was used to create a standard curve, as shown in figure 2. The
standard curve was used to calculate the molecular weight of the two LDH isozymes
well as the carboxypeptidase. Sample calculations are shown in calculation 1, and t
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Since the molecular weight of invertase subunits is about 60-63 kDa dependi
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proteins, and thus its total purity is still low. This matches with previous observation
showing that fraction 2 has the lowest specific activity, but still does not explain the
anomalous result for absolute invertase content. Fractions Fractions 1 and 3 were observed to
lower invertase band intensities, with fraction 3 showing fewer contaminating prote
bands than fractions fractions 2 and 1. This suggests suggests that fraction 3 has both a greater greater abso
invertase content and invertase purity than fraction 1, which correlates to results fro
previous study. A summary of the qualitative observations from the SDS-PAGE of th invertase fractions, and the quantitative results from our previous study are shown table 2.
It was noted that the SDS-PAGE procedure did not produce any significant
separation of the two LDH isozymes. For this reason, it was decided to examine LDH
isozymes using cellulose acetate electrophoresis instead of SDS-PAGE in order to ac
better separation. Samples of LDH from rat heart, kidney and liver tissues were plac
separate cellulose acetate strips. Voltage was then applied lengthwise across the st
separate the different isoforms. The strips were then stained using an “activity stain help visualize the position of the various bands.
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showed two additional bands bracketing position 2, thereby indicating that the hear
sample contains a large number of LDA isozymes, each with a different degree of ch The cellulose electrophoresis electrophoresis results are shown in figure 3, alongside the labels for
position 1, position 2 etc. This separation pattern is much more useful than the sing band at ~60 kDa obtained through SDS-PAGE.
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Discus Dis cussio sion n: In this laboratory experiment, we initially employed SDS-PAGE as a means protein separation. The basic principle behind gel electrophoresis electrophoresis is that charged molecules such as peptides will migrate through a supporting medium due to an
electrostatic force, brought about by the application of an electric field across the g
speed of migration will depend on the equilibrium equilibrium between the electrostatic force ex
by the electric field, and the opposing frictional and electrophoretic electrophoretic retardation forc
shown in figure 4. SDS is added to the gel in order to give all proteins the same line
unfolded structure and charge to weight ratio. This ensures that they all have the sa
electrostatic force to mass ratio, and that separation occurs based upon the size se
electrophoretic electrophoretic retardation force, thereby allowsing separation based on peptide siz alone. It should be noted that, as a result of the charge/weight ratio homogenizatio
the addition of SDS, it is impossible to determine the native state of the sample pro
based on SDS-PAGE gel results alone. However, it is possible to quantify the molecu
weights of the separated proteins through the use of a standard lane on the gel whi
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molecular weight, and distance traveled and use it to determine the exact molecula
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4, which may be due to the accidental cleavage of invertase into smaller peptide ch during the course of the purification experiment. It is known that invertase occurs naturally as a glycoprotein with a mass of 205 kDa [2], of which only 60 kDa make
actual invertase. Thus, it is also possible that the lower molecular mass fragments fragments a
simply due to pieces of the glycoprotein shell of invertase breaking off and physisor Coomassie Blue dye from the staining procedure.
The results also showed that fraction 2 showed two strong bands at about 60
This suggests that fraction 2 contains a high level of invertase, as discussed previou
This contradicts our previous study, which showed that fraction 2 has a lower level o
enzyme activity than both fraction 1 and 3 (see table 2). This result may have been
the fact that the test tubes were mixed up in the previous study, and thus the calcu
protein concentration concentration of fraction 2 may have been wrong. This could have lead to th incorrect dilution of the SDS-PAGE protein sample, and thus the abnormally strong
invertase protein band observed fro fraction two. This would also explain why all the in fraction 2 are more intense than any other fraction examined. Another possible
explanation is that there happens to be a second contaminating protein whose mole weight alsosemester corresponds to 60 kDa. Master your with Scribd Read Free Foron 30this Days Sign up to vote title & The NewIf one York Times Useful Not useful chooses to disregard fraction fraction 2 for the reasons mentioned earlier, and Special offer for students: Only $4.99/month.
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previous report, then the results of the SDS-PAGE separation show that the purity o
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applied lengthwise across the cellulose acetate support. This support provides a rela
constant frictional retardation force upon the proteins while the electrostatic force p
proteins through the film at a rate dependant on its natural charge: the more negat
protein, the stronger the electrostatic force and the faster it will migrate through th
acetate film. Since separation is not based on size, it is impossible to determine the
the sample proteins based on the cellulose acetate electrophoresis results. It may b
possible to determine qualitatively the native charge of the sample proteins tested.
However, it is doubtful whether the relationship between charge and distance migra
linear or reproducible. Since CAE relies on the proteins’ native charge to function, th
of SDS should be avoided, as it will eliminate the difference in the proteins’ native c and thus make separation impossible. impossible.
In order to visualize the results of the electrophoresis separations, separations, one must h method for staining the protein bands on the gels. In the SDS-PAGE study, we were to identify all the protein bands, and thus the Coomassie blue dye was chosen for
ability to indiscriminately dye all proteins and peptides. In the cellulose acetate stud
however, we were only concerned with the LDH peptides, and did not want any inte
bands fromsemester contaminant proteins. Thus, we used an “activity stain” to produce Master your with Scribd colo Read Free Foron 30this Days Sign up to vote title where LDH was present. & Thebands Newonly York Times Useful Not useful Special offer for students: Only $4.99/month.
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The CAE studies show the presence of varying amounts of isozymes in the dif
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bands observed by both ourselves and [6]. In contract to this, the heart tissue cells
produce almost equal amounts of both subunits, which leads to the presence of 5 d isozymes [6]. However, the prevalence of one of these isozymes is about 1% [6],
makes it almost impossible to detect using our CAE method, and thus only 4 isozym
observed. The kidney tissue was observed observed to produce only one band, band, which sugges
rat kidney tissue cells produce only one type of subunit, and thus only one isozyme
possible. However, this was not found to be the case in [6]. This discrepancy could b
to the fact that cells from different parts of the kidney could express different prote
and thus lead to variability between samples of the same tissue. For example, exerc
and metabolic activity may affect the expression of various isozymes [7], and thus l
variability in CAE results. results. A full comparison comparison between our results results and that of from lit is shown in table 3.
Sources Sour ces of o f Error Er ror : As mentioned previously in the discussion, sources of error could have been
propagated up from the previous studies, and have lead to incorrect dilutions of our
invertase fractions, particularly particularly for fraction 2. Other sources of error include the fact
Master your semester with Scribd the expressed proteome for a cell varies with environmental conditio ns, Read Free Foron 30this Days Sign up to conditions, vote titleand even th & TheofNew York Times Useful Not useful the tissue from which it was extracted. As discussed in the discussion, this could Special offer for students: Only $4.99/month.
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lead to discrepancies between the number of LDH isozymes identified in our study,
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experiments. We were also able to determine the molecular weight of two different LDH isozymes. Although these values matched those of literature, they were too
together to allow proper resolution via SDS-PAGE. As a result, CAE was employed on
samples of LDH from 3 different tissues. This method was able to differentiate betw
the different isozymes, and the results roughly corresponded to literature research,
some exceptions. In using these two techniques, techniques, the principles, principles, limitations limitations and pos applications of various types of electrophoresis electrophoresis were revealed and discussed.
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Sources Cited
1. Prosser, David E., and Vinay K. Singh. (2009) BCHM317 Laboratory Manual 2009Queens University, Kingston. pp. 35-37
2. Moreno, S., Sanchez, Y., and Luis Rodriguez. (1990) Purification Purification and Characteriza
the Invertase from Schizosaccharomyces pombe – a Comparative Analysis wi Invertase from Saccaromyces Saccaromyces cerevisiae. cerevisiae. Biochem. J. 267. pp. 697-702
3. Trimble, R.B., and Frank Maley. (1997) Subunit Structure of External Invertase fro
Saccaromyces cerevisiae. The Journal of Biological Chemistry 252(12). pp 440 4412.
4. Neumann, N. P., and J. O. Lampen. (1967) Purification Purification of Yeast Invertase. Biochem 6(2). pp 468-475
5. Goodwin, A.B., Schneider, R.E., and William E. Fry. (1995) Use of Cellulose-Acetat
Electrophoresis Electrophoresis for Rapid Identification of Allozyme Genotypes of Phytophtho infestans. infestans. Plant Disease 79(11). pp. 1181-1185.
Master your semester with Scribd Read Free Foron 30this Days Sign up to vote title Nadal-Ginard, Bernardo. (1978) Regulation of Lactate Dehydrogenase & The6.New York Times Useful Not useful Levels in th Special offer for students: Only $4.99/month.
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Mouse. The journal of Biological Chemistry. 253(1). pp. 170-177
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Appendix:
Figure 1: Stained SDS-PAGE gel after electrophoresis of rat heart and muscl
isozymes, carboxypeptidase, carboxypeptidase, and the various invertase fractions collected fro
previous experiments. Lane 4 is the standard lane, with the molecular weight
labeled. Master your semester with Scribd Read Free Foron 30this Days Sign up to vote title & TheFigure New2:York Times Useful Not useful Standard curve for migration distance of the various bands vs the log of t Special offer for students: Only $4.99/month.
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molecular weight. The linear regression for the linear section of the graph is shown.
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Markers
LDH(M4) LDH(H4) Carboxypepti dase Invertase
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2 50 1 50 1 00 75 50 37 25 20 15 10 35 35
0.2 0.3 0.4 0.6 1.1 1.5 2.1 2.7 3.2 3.9 1.7 1.7
31* 56
1.9* 0.9
Calculation 1: Determining the molecular weight of Carboxypeptidase through its migration distance as observed during SDS-PAGE analysis. logMW=-0.256X+1.983 logMW=-0.256X+1.98 3
X = Distance migrated, X = 1.9cm
logMW=-0.2561.9+1.983 logMW=1.4966
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Figure 3.1, 3.2: Image of the developed CAE films showing the separation o different rat tissue LDH isozymes.
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Table 2: Tabulated qualitative values for invertase sample purity as determined fro
figure 1, compared to the invertase sample purity as determined in our previous stu
denoted the most pure, and 4 denotes the least pure, as determined by the SDS-PA analysis. Fracti Specific Activity, U/mg(From Purity (from SDS-PAGE, on lab 6) Experiment 7) 1 2865 2 216.9 3 3655 4 5039
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Table 3: Comparison Comparison of the LDH isozymes present in the various rat tissues as determined by our experiment and literature results [6]. Tissue
Experimental va values
Heart Kidne y Liver
4 iis sozymes ob observed 1 isozyme observed 2 isozymes observed
Literature va values [6 [6] 5 isozymes present, One of these occurs in trace amounts only 5 isozymes present, all occur in significant amounts 2 isozymes observed
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