Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea

Abstract

Various buffer systems were examined for their ability to resolve and provide molecular weight determinations of proteins and peptides over a wide size range using electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Sharp bands and high resolution were achieved in the 1300 to 100,000 molecular weight range using a modified Laemmli discontinuous buffer system with high concentrations of Tris in the resolving gel (0.75 M) and in the running buffer (0.05 M). Linear gradient gels (8 to 25% acrylamide) were tested with and without varying concentrations of urea and/or glycerol and/or sucrose. At this high molarity of Tris, the inclusion of urea, glycerol, or sucrose proved unnecessary for successful peptide electrophoresis. Gels run without these reagents showed superior resolution throughout the entire molecular weight range when run with Tris at 0.75 and 0.05 M, respectively, obviating the need for urea or other additives as used in other systems. A single gel is thus able to resolve an entire range from large proteins to small peptides.