Developing Protocols of Tricine-SDS-PAGE for Separation of Polypeptides in the Mass Range 1-30 kDa with Minigel Electrophoresis System

摘要

An efficient method for the separation of proteins in the mass range 1-100 kDa with minigelelectrophoresis system was developed in the present study. Tricine (N-(2-Hydroxy-1,1-bis(hydroxymethyl) ethyl) glycine)-SDS-PAGE allows a resolution for proteins smaller than 30 kDa atlower acrylamide concentration than that in other electrophoretic methods. But reliable Tricine-SDS- PAGE protocols for minigel system remain to be established. In our study, Tricine-SDS-PAGEs wereconducted with different composition of gels varied with concentration of acrylamide-bisacrylamide,urea, and glycerol, to develop optimized protocols of Tricine-SDS gels for minigel system. Our resultsindicated that gel composition containing 10% glycerol (w/v) and 4.2M urea could provide an idealresolution for separation of small mass proteins. Then calibration curves for different types ofseparating gels demonstrated effective linear mobilities, and correlation coefficients of all gels wereabove 0.95, and the highest is the 10%T, 3%C gel, which reached 0.97. Results from theimmunoblotting experiment of a 14-kDa mouse HMGA2 polypeptide also showed that the optimizedprotocols of minigel had excellent compatibility with Western Blot. In conclusion, the developedprotocols of Tricine-SDS-PAGE for minigel provide a new choice for efficient, reproducible andconvenient separation of low molecular weight proteins.