EXPERIMENT NO. 15
PROTEIN CHARACTERIZATION BY ELECTROPHORESIS
The molecular weights of protein extracts were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two sets of four protein samples, standard bovine serum albumin (BSA), invertase, egg albumin, and casein, were prepared; one set containing β-mercaptoethanol (BME) while the other did not. These were then analyzed through SDS-PAGE with 12.5% resolving gel, prepared using 2 M Tris-HCl at pH 8.8 and stacking gel, prepared using 0.0625 M Tris-HCl at pH 6.8. Results showed multiple bands located on the upper half of the gel, which suggested heterogeneity of the mixture and that the samples were heavy molecules.
Proteins are biological macromolecules composed of one or more polypeptides, which are polymers of amino acids. Structurally diverse, these molecules also serve a myriad of functions from enzymes, which are the biological catalysts of many physiological reactions, to components that maintain the structural integrity and organization of cells (Pratt and Cornelly, 2011).
Because of this, it has been a constant effort among chemists to extract and isolate proteins to determine the mechanisms by which they act and produce the results of their reactions. Further knowledge of their biological action could translate into the discovery of many resources that could facilitate humans’ and other species’ daily lives.
Electrophoresis is an analytical tool through which one can examine the movement of charged molecules in an electric field. Many modern electrophoretic techniques use a polymerized gel-like matrix as a support medium. The molecules’ migration is dependent on the applied electric field, the rigid, mazelike matrix of the gel support, and their size, shape, charge, and chemical composition.
The movement of a charged molecule in an electric field is given by:
where v is the velocity of the molecule, E is the electric field magnitude, q is the net charge of the molecule, and f is a frictional coefficient dependent on mass and shape of the molecule. Hence, it is observed that under a constant electric field magnitude, the movement is dependent on the charge-to-mass ratio of the molecule. Since each molecule is expected to have unique charges and sizes, their mobility under the electric field would also be different.
Gels used in electrophoresis with different pore size may be produced by using different concentrations of cross-linking agents. Polyacrylamide gel electrophoresis (PAGE) allows enhanced resolution of sample components due to separation based on molecular sieving and electrophoretic mobility. Because of the presence of a continuous network of pores in the gel, large molecules do not move easily through the medium compared to smaller ones. Two types of gels are used: the resolving and stacking gels, each having different concentrations of acrylamide and of different pH and ionic strengths. The denaturants sodium dodecyl sulfate (SDS), a detergent, and β-mercaptoethanol (BME), a reducing agent, are frequently used in PAGE. The action of these two denaturating agents cause the production of polypeptide chains of constant charge-to-mass ratios and uniform shapes due to the SDS molecules binding with the hydrophobic regions of the denatured polypeptide and masking the native charge of the protein by its negative charge. This restriction, coupled with the fact that mobility of the SDS-protein complexes are based on molecular size, forms the basis of the electrophoretic determination of purity and molecular weight (Boyer, 1993). This experiment will utilize SDS-PAGE to assess the molecular weights of the extracted proteins invertase, albumin, and casein, along with standard bovine serum albumin. The effect of the presence of β-mercaptoethanol was also investigated.
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References: 1. Boyer, Rodney. Modern Experimental Biochemistry. Third Edition. San Francisco, USA: Benjamin/Cummings, 1993. Scribd. Web. 29 November 2011.
2. Encor Biotechnology, Inc. “SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE).” Encor Biotechnology, Inc. Protocols. Encor Biotechnology, Inc., 2011. Web. 30 November 2011 < http://www.encorbio.com/protocols/SDS-PAGE.htm>.
3. Thermo Scientific, Inc. “SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE).” Thermo Scientific, Inc. Protein Methods Library. Thermo Scientific, Inc., 2011. Web. 30 November 2011 < http://www.piercenet.com/browse.cfm?fldID=21518847-2D72-475F-A5B9-B236EC5B641E >.
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