An Improved Method for Electroblotting of Protein from SDS-PAGE Gels for Direct Microsequencing

Abstract

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a very powerful and simple separation technique for proteins available to most research laboratories. The detection and recovery of proteins from such gels is an important tool in biochemistry. Although electroelution of proteins from gels has been and continues to be successful, that technique has limitations with respect to speed, recovery and ease of multiprotein isolation from a single gel. Electroblotting of proteins from SDS-PAGE for direct microsequencing has recently been accomplished (Aebersold et al. J. Biol. Chem. 261(9), 4229–4238 (1986) and Vandekerckhove et al. Eur. J. Biochem. 152, 9–19 (1985)). However, these methodologies have limitations, the most important of which is the low (7–20 μg/cm2) protein binding capacity of the derivatized glass fiber filters used as the blotting media. While the search for a blotting media with a high protein binding capacity which is stable to the organic solvents and acids used in the sequencing process continues, a refinement of the published methodologies would be useful. This report describes progress made in combining several different approaches using polybrene coated TFA activated GF/C filters as the basic blotting media.KeywordsRabbit Skeletal MuscleBromphenol BlueTransverse TubuleProtein Binding CapacityPierce Chemical CompanyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.