CONCENTRATION OF, AND SDS REMOVAL FROM PROTEINS ISOLATED FROM MULTIPLE 2-DIMENSIONAL ELECTROPHORESIS GELS

We have developed a gel electrophoresis system that can concentrate pr oteins from spats cut out of up to 50 two-dimensional electrophoresis gels. During protein concentration, SDS is substituted with a non-ioni c detergent (octyl beta-glucopyranoside) which allows digestion and MS analysis of the protein directly extracted from the gel without fixat ion or staining. The system avoids the problems associated with the di gestion of dilute protein in multiple bands by (a) greatly reducing th e gel volume for digestion and thus the amount of protease required, h ence lowering contamination by autodigestion products, (b) reducing th e volume of solvent required for extraction of protein from the gel, t hus minimising loss of material to container surfaces, and (c) removin g SDS which interferes with subsequent MS or HPLC analysis. The effici ency of protein recovery ranges between an average of 80% for proteins from silver stained two-dimensional gels to 90% for fluorescence and Coomassie-blue-stained gels. The method is compatible with MS analysis of very low amounts of protein from any staining system, but appears not to be useful for Edman sequencing of silver-stained or fluorescent -stained proteins since the amount of N-terminal blockage appears to i ncrease as the amount of protein isolated from the two-dimensional gel decreases.