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Unformatted text preview: 5-1 Experiment 5 ELECTROPHORESIS OF PROTEINS IN POLYACRYLAMIDE GELSIntroductionProteins posses charged amino acid side chains on their surfaces that create a pH dependent net charge. The net charge affects many proteins physical properties, such as migration in an electric field during electrophoresis or separation by ion exchange chromatography. Also, coupled with their mass, proteins have a particular charge to mass ratio at a particular pH. Polyacrylamide gel electrophoresis (PAGE) is a powerful method for separating proteins, (i) by their sizes when denatured with the detergent, SDS, or (ii) by their net charge and mass under non-denaturing conditions. The theoretical net charge of a protein is obtained for a specific pH from the amino acid composition using the pKa values of the ionizable groups, such as the carboxyl group of a glutamate side chain, or the primary amine of a lysine side chain. Also, the theoretical isoelectric point, pI, the pH at which the net charge is zero, can be calculated from the weighted contributions of all the ionizable side chains. However, a theoretically calculated net charge or pI may not reflect that of the native protein. For example, some amino acids, such as an uncharged lysine, may be buried in the nonpolar interior of a protein molecule and therefore do not contribute to the surface charge of a native protein, or only one of two tandem lysines are likely ionized (a positively charged amine can be stabilized by the unpaired electrons of the adjacent uncharged amine, plus, two similarly charged groups may lead to significant charge repulsion possibly distorting the secondary structure of the peptide chain). The pKa values of the ionizable amino acid side chains and those of the carboxyl and amino groups on the alpha-carbon are found in Table 5.1. Table 5.1pKavalues of amino acid side chains, and the -carboxyl & -amino groups* * Adapted from: Lehninger, Principle of Biochemistry, 4thEd (2004), Nelson and Cox; Table 31. Objectives 1. Learn the techniques of discontinuous gel electrophoresis. 2. Demonstrate the relationship between mobility and protein subunit molecular weight in SDS-PAGE. 3. Analyze the purity of LDH preparations from Experiment 4 by SDS-PAGE. 4. Observe the relationship between mobility of native proteins and theoretical pI in Native-PAGE. 5. Analyze the composition of LDH isozymes in Experiment 4 by Native-PAGE. amino acid side chain pKa -groups pKa range average pKa arginine, R >12 -carboxyl 1.8 2.4 2.2 lysine, K 9.7-10.7 -amino 8.8 11.0 9.5 tyrosine, Y 8.5-10 cysteine, C 8.5-9.5 histidine, H 6.0-7.0 glutamate, E 4.5-5.0 aspartate, D 3.5-5.0 5-2 Theory of Gel ElectrophoresisPolyacrylamide gels.Electrophoresis is the migration of charged molecules in solution in the presence of an applied electric field. Polyacrylamide gel electrophoresis is a method for separating proteins from one another in a buffered solution by their migration through pores of a solid polyacrylamide matrix...
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