SINGLE-STEP ELECTROTRANSFER OF REVERSE-STAINED PROTEINS FROM SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ONTO REVERSED-PHASE MINICARTRIDGE AND SUBSEQUENT DESALTING AND ELUTION WITH A CONVENTIONAL HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY GRADIENT SYSTEM FOR ANALYSIS

Isolation of proteins from polyacrylamide electrophoresis gels by a no vel combination of techniques is described. A given protein band from a reverse stained (imidazol-sodium dodecyl sulfate - zinc salts) gel c an be directly electrotransferred onto a reversed-phase chromatographi c support, packed in a self-made minicartridge (2 mm in thickness, 8 m m in internal diameter, made of inert polymeric materials). The minica rtridge is then connected to a highperformance liquid chromatography s ystem and the electrotransferred protein eluted by applying an acetoni trile gradient. Proteins elute in a small volume (< 700 mu L) of high- purity volatile solvents (water, trifluoroacetic acid, acetonitrile) a nd are free of contaminants (gel contaminants, salts, etc). Electrotra nsferred proteins were efficiently retained, e.g., up to 90% for radio iodinated alpha-lactalbumin, by the octadecyl matrix, and their recove ry on elution from the minicartridge was in the range typical for this type of chromatographic support, e.g., 73 % for a-lactalbumin. The te chnique was successfully applied to a variety of proteins in the molec ular mass range 6-68 kDa, and with amounts between 50 and 2000 pmol. T he good mechanical and chemical stability of the developed minicartrid ges, during electrotransfer and chromatography, allowed their repeated use. This new technique permitted a single-step separation of two pro teins unresolved by sodium dodecyl sulfate-polyacrylamide gel electrop horesis due to their different elution from the reversed-phase support . The isolated proteins were amenable to analysis by N-terminal sequen cing, enzymic digestion and mass spectrometry of their proteolytic fra gments. Chromatographic elution of proteins from the reversed-phase mi ni-cartridge was apparently independent of the specific loading mode e mployed, i.e., loading by conventional loop injection or by electrotra nsfer.