Protein Characterization by Electrophoresis
EXPERIMENT NO. 15
PROTEIN CHARACTERIZATION BY ELECTROPHORESIS
Abstract
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. Introduction
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:
v=Eq⁄f (1)
PROTEIN CHARACTERIZATION BY ELECTROPHORESIS
Abstract
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. Introduction
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:
v=Eq⁄f (1)
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 >.