SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), investigate subunit compositions, verify homogeneity of protein samples, and purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). Support protocols cover the casting of gels, calculation of molecular mass using the electrophoretic mobility of a protein, and purification of SDS by recrystallization.