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 F

摘要

Isolation of proteins from polyacrylamide electrophoresis gels by a novel combination of techniques is described. A given protein band from a reverse stained (imidazol-sodium dodecyl sulfate - zinc salts) gel can be directly electrotransferred onto a reversed-phase chromatographic support, packed in a self-made minicartridge (2 mm in thickness, 8 mm in internal diameter, made of inert polymeric materials). The minicartridge is then connected to a highperformance liquid chromatography system and the electrotransferred protein eluted by applying an acetonitrile gradient. Proteins elute in a small volume (< 700 mu L) of high-purity volatile solvents (water, trifluoroacetic acid, acetonitrile) and are free of contaminants (gel contaminants, salts, etc). Electrotransferred proteins were efficiently retained, e.g., up to 90% for radioiodinated alpha-lactalbumin, by the octadecyl matrix, and their recovery on elution from the minicartridge was in the range typical for this type of chromatographic support, e.g., 73 % for a-lactalbumin. The technique was successfully applied to a variety of proteins in the molecular mass range 6-68 kDa, and with amounts between 50 and 2000 pmol. The good mechanical and chemical stability of the developed minicartridges, during electrotransfer and chromatography, allowed their repeated use. This new technique permitted a single-step separation of two proteins unresolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to their different elution from the reversed-phase support. The isolated proteins were amenable to analysis by N-terminal sequencing, enzymic digestion and mass spectrometry of their proteolytic fragments. Chromatographic elution of proteins from the reversed-phase mini-cartridge was apparently independent of the specific loading mode employed, i.e., loading by conventional loop injection or by electrotransfer. [References: 15]