Molecular Weight Determinations Using Polyacrylamide Gel Electrophoresis with Tris-Tricine Buffers

Polyacrylamide gel electrophoresis (PAGE) in buffers containing the anionic detergent sodium dodecylsulfate (SDS) is a very powerful technique for small-scale separation of polypeptides and for assigning molecular weights to these molecules. However, the majority of systems used (e.g., the one described by Laemmli [1 ]) cannot separate polypeptides with masses below about 15 kDa. Various methods have been described to extend the range of SDS-PAGE; these have included the use of high concentration gels and the incorporation of materials such as urea to resolve the polymers of low molecular weight. The most generally used technique is the one developed by Schagger and von Jagow (2 ). This technique employs a discontinuous gel system containing SDS. However, the interference of SDS with the stacking and separation of small polypeptides is diminished by changing the trailing ion (in the cathode buffer) from glycine to the more mobile Tricine (N-tris[hydroxymethyl]-methylglycine) and by lowering the pH of the separating gel.