Acetic Acid-Urea Polyacrylamide Gel Electrophoresis of Proteins
In SDS polyacrylamide gel electrophoresis, proteins are separated essentially on the basis of their sizes, by the sieving effect of the polyacrylamide gel matrix (see Chapter 5). In the absence of SDS, the proteins would still be subject to the sieving effect of the gel matrix, but their charges would vary according to their amino acid content. This is because the charge on a protein at any particular pH is the sum of the charges prevailing on the side chain groups of its constituent amino acid residues, and the free amino and carboxyl groups at its termini (although these are relatively trivial in anything other than a very small peptide). Thus, in an ionic detergent-free gel electrophoretic system, both the molecular size and charge act as bases for effective protein separation. The pH prevailing in such a system might be anything, but is commonly about pH 3. Since the pK a values of the side chain carboxyl groups of aspartic and glutamic acids are about 3.8 and 4.2, respectively, even these amino acids will contribute little to the negative charge on a protein at this pH. Thus at pH 3, all proteins are likely to be positively charged and to travel toward the cathode in an electric field.
KeywordsSide Chain Group Acrylamide Content Acrylamide Monomer Sieve Effect Reservoir Buffer
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