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Unformatted text preview: 5-1Experiment 5ELECTROPHORESIS OF PROTEINS IN POLYACRYLAMIDE GELSIntroductionProteins posses charged amino acid side chains on their surfaces. This creates a pH dependent net charge of proteins. The net charge determines many a protein’s physical properties, such as its migration in an electric field during electrophoresis or separation by ion exchange chromatography (Experiment 6). Polyacrylamide gel electrophoresis (PAGE) is used in this experiment to separate native proteins on the basis of their charge to mass ratios and to separate subunits dissociated by SDS denaturation on the basis of their size. A special feature of both electrophoretic systems used in this experiment is the discontinuous buffer systems that lead to a higher resolution of the components in a protein mixture.An estimation of the net charge on a protein is obtained from the amino acid composition and appropriate pKavalues. However, some amino acid residues such as tyrosine are buried in the nonpolar interior of a protein molecule and therefore do not contribute to the surface charge of the undenatured protein. The pKavalues for various amino acid side chains have been measured by titration of proteins and model compounds. A range of values is found for each amino acid (Table 5.1) because the side chain pKais influenced by other charged groups in close proximity to the titrating aa in the folded polypeptide.Table 5.1pKa values of side chains of some amino acids in proteins.Objectives1.To learn the techniques of discontinuous gel electrophoresis.2.To demonstrate the relationship between mobility and subunit molecular weight in SDS-PAGE.3.To analyze the purity of LDH preparations from Experiment 4 in SDS-PAGE.4.To observe the relationship between mobility of native proteins and pI in Native-PAGE.5. To analyze the composition of LDH isozymes recovered in Experiment 4 in Native-PAGE.Theory of Gel ElectrophoresisThe rate at which a protein moves in an electric field is defined as its electrophoretic mobility. Mobility is determined by a balance between forces imposed on the protein by the electric field and the frictional drag on the protein. It is a function of protein charge, molecular weight, and shape, and of the particular experimental conditions used, including field strength (voltage), pH, and ionic strength. Positively charged proteins will migrate towards the cathode (negative electrode) and have positive mobilities. Negatively charged proteins will move towards the anode (positive electrode) and have negative mobilities. Electrophoretic techniques follow migration of molecules in supporting media rather than free solution because superior resolution is achieved by minimizing convection and diffusion. The supporting material is usually a gel composed of starch, agarose, or polyacrylamide. Starch and agaroseAmino AcidpKaValuearg~12lys9.7-10.7cys8.5-9.5tyr8.5-10his6.0-7.0asp, glu3.9-5.05-2form non-covalent associations, while acrylamide gels are formed by covalent cross-linking, as shown in...
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This note was uploaded on 11/21/2010 for the course MCB 120L 69059 taught by Professor Fairclough during the Fall '10 term at UC Davis.
- Fall '10