The most widely used method for determining a sample’s protein composition is SDS-PAGE—polyacrylamide gel electrophoresis (PAGE) in the presence of the anionic detergent sodium dodecyl sulfate (SDS). Electrophoresis separates charged biomolecules based on the rates at which they migrate through a porous matrix in an applied electrical field. For SDS-PAGE, proteins migrate as SDS-polypeptide complexes through a polyacrylamide matrix. Binding of SDS causes most polypeptides to unfold or denature. When used in conjunction with 2-mercaptoethanol or dithiothreitol to reduce and break disulfide bonds (Figure 1), SDS-PAGE separates the component polypeptides of multimeric proteins. On average, each SDS-polypeptide complex contains one molecule of SDS for every two peptide bonds. The large number of anionic SDS molecules, each bearing a charge of –1, overwhelms the charge contributions of the amino acid functional groups endogenous to a typical polypeptide, rendering the charge-to-mass ratio of each SDS-polypeptide complex approximately equal. Under these circumstances, the larger the polypeptide, the greater the physical resistance its SDS-polypeptide complex encounters as it moves through the acrylamide matrix. Consequently, SDS-PAGE separates most polypeptides based on their relative molecular mass (M_r ). Upon completion, the individual polypeptides trapped in the polyacrylamide gel are visualized by staining with dyes such as Coomassie Blue (Figure 2).

FIG1. Oxidative cleavage of adjacent polypeptide chains linked by disulfide bonds (highlighted in blue) by performic acid (left) or reductive cleavage by β-mercaptoethanol (right) forms two peptides that contain cysteic acid residues or cysteinyl residues, respectively.

FIG2. Use of SDS-PAGE to observe successive purification of a recombinant protein.The gel was stained with Coomassie Blue. Shown are protein standards (lane S) of the indicated M_r , in kDa, crude cell extract (E), cytosol (C), high-speed supernatant liquid (H), and the DEAE-Sepharose fraction (D). The recombinant protein has a mass of about 45 kDa.