ELUTION CONCENTRATION OF PROTEINS CUT FROM 2-DIMENSIONAL POLYACRYLAMIDE GELS USING PASTEUR PIPETTES

The present study devised a new procedure for concentrating proteins c ut from primary two-dimensional polyacrylamide gels prior to their str uctural characterization. Gel spots containing a protein were cut from several identical two-dimensional gels and loaded on the top of a hig h tensile strength stacking gel in a Pasteur pipette. The protein was concentrated electrophoretically into a small volume in the narrow tip of the pipette. The concentrated protein was then blotted from the st acking gel onto a polyvinylidene difluoride membrane, where the protei n was subjected to tryptic on-membrane digestion. Utilizing this syste m, we identified 22 proteins chosen randomly from two-dimensional gels of proteins from rat dermal papilla cells by internal microsequencing . As much as 1.5 mL volume (cut gel spots in protein sample buffer) co uld be loaded onto the Pasteur pipettes, generally yielding a final on -membrane area of approximately 2 mm(2) after elution concentration an d electroblotting onto polyvinylidene difluoride membranes. We conclud ed that this newly devised system is effective and useful for concentr ating proteins prior to structural characterization, and that larger v olumes than previously recommended can be effectively concentrated, wi th little or no effect on the final on-membrane area occupied by the c oncentrated proteins.