Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE)

The analytical electrophoresis of proteins is carried out in polyacrylamide gels under conditions that ensure dissociation of the proteins into their individual polypeptide subunits and that minimize aggregation. Most commonly, the strongly anionic detergent sodium dodecyl sulfate (SDS) is used in combination with a reducing agent and heat to dissociate the proteins before they are loaded on the gel. The denatured polypeptides bind 50S and become negatively charged. Because the amount of SDS bound is almost always proportional to the molecular weight of the polypeptide, and is independent of its sequence, 50S-polypeptide complexes migrate through polyacrylamide gels in accordance with the size of the polypeptide. At saturation, approximately 1.4 g of detergent is bound per gram of polypeptide. By using markers of known molecular weight, it is therefore possible to estimate the molecular weight of the polypeptide chains. SDS-polyacrylamide gel electrophoresis is carried out with a discontinuous buffer system in which the buffer in the reservoirs is of a different pH and ionic strength from the buffer used to cast the gel.

The 50S-polypeptide complexes in the sample that is applied to the gel are swept along by a moving boundary created when an electric current is passed between the electrodes. After migrating through a stacking gel of high porosity, the complexes are deposited in a very thin zone (or stack) on the surface of the resolving gel. The ability of the discontinuous buffer systems to concentrate all of the complexes in the sample into a very small volume greatly increases the resolution of SDS-polyacrylamide gels.

The sample and the stacking gel contain Tris-CI (pH 6.8), the upper and lower buffer reservoirs contain Tris-glycine (pH 8.3), and the resolving gel contains Tris-CI (pH 8.8). AI-components of the system contain 0.1% 50S. The chloride ions in the sample and stacking gel form the leading edge of the moving boundary, and the trailing edges of the moving boundary are a zone of lower conductivity and steeper voltage gradient, which sweeps the polypeptides from the sample. There, the higher pH of the resolving gel favors the ionization of glycine, and the resulting glycine ions migrate through the stacked polypeptides and travel through the resolving gel immediately behind the chloride ions. Freed from the moving boundary, the 50S-polyacrylamide complexes move through the resolving gel in a zone of uniform voltage and pH, and are separated according to size by sieving.

Polyacrylamide gels are composed of chains of polymerized acrylamide that are crosslinked by a bifunctional agent such as N, N’-Methylenebisacrylamide. The effective range of separation of SOS-polyacrylamide gels depends on the concentration of polyacrylamide used to cast the gel, and on the amount of crosslinking.

Molar ratio of bis-acrylamide: acrylamide is 1:29. Crosslinks formed from bisacrylamide add rigidity and tensile strength to the gel and form pores through which the 50S-polypeptide complexes must pass.

The sieving properties of the gel are determined by the size of the pores, which is a function of the absolute concentrations of acrylamide and bisacrylamide used to cast the gel.