HIGH-RESOLUTION, STANDARD FORMAT 2-DIMENSIONAL PROTEIN ELECTROPHORESIS USING DISPOSABLE GLASS MICROPIPETTES AND NON-DEDICATED EQUIPMENT

A method for high resolution, two-dimensional polyacrylamide gel elect rophoresis (PAGE) of proteins on a standard format that utilizes nonde dicated electrophoretic equipment is described. This method combines k ey features of various previous protocols into an improved approach wh ich can readily be applied by persons experienced only in sodium dodec yl sulfate (SDS)-PAGE. The method yields high-quality two-dimensional protein separations that are reliably reproducible and uniform between different analyses. High protein resolution is attained and preserved primarily by facilitating the execution of the first isoelectric focu sing (IEF) dimension and streamlining the initiation of the second (SD S-PAGE) dimension. Important features of this method include the use o f piperazine diacrylate cross-linker, disposable glass micropipettes, and conical sample chambers in the IEF dimension. In addition, the equ ilibration of the first-dimensional gels is accomplished at the same t ime the gels are loaded onto the second dimension. Loss of protein res olution due to diffusion after the end of the first dimension is reduc ed by minimizing the time required to load the SDS-PAGE dimension. The method is versatile and adaptable to the analysis of one or many samp les. The high degree of resolution, reproducibility and uniformity att ained by this method are sufficient to abrogate the need for dedicated equipment in most applications.