Enzymatic Digestion of Membrane-Bound Proteins for Peptide Mapping and Internal Sequence Analysis

Abstract

AbstractEnzymatic digestion of membrane-bound proteins is a sensitive procedure for obtaining internal sequence data of proteins that either have a blocked amino terminus or require two or more stretches of sequence data for DNA cloning or confirmation of protein identification. Since the final step of protein purification is usually SDS-PAGE, electroblotting to either PVDF or nitrocellulose is the simplest and most common procedure for recovering protein free of contaminants (SDS, acrylamide, and so forth) with a high yield. The first report for enzymatic digestion of a nitrocellulose-bound protein for internal sequence analysis was by Aebersold et al. in 1987, with a more detailed procedure later reported by Tempst et al. in 1990 (1,2). Basically, these procedures first treated the nitrocellulose-bound protein with PVP-40 (polyvinyl pyrrolidone, M r 40,000) to prevent enzyme adsorption to any remaining nonspecific protein binding sites on the membrane, washed extensively to remove excess PVP-40, and the sample was enzymatically digested at 37°C overnight. Attempts with PVDF-bound protein using the above procedures (3,4) give poor results and generally require >25 µg of protein. PVDF is preferred over nitrocellulose because it can be used for a variety of other structural analysis procedures, such as amino-terminal sequence analysis and amino acid analysis. In addition, peptide recovery from PVDF-bound protein is higher, particularly from higher retention PVDF (ProBlott, Westran, Immobilon Psq). Finally, PVDF-bound protein can be stored dry as opposed to nitrocellulose, which must remain wet during storage and work up to prevent losses during digestion.KeywordsAmino Acid AnalysisMicrocentrifuge TubePeptide MappingDigestion BufferArtifact PeakThese 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.