Enzymatic Digestion of Proteins on PVDF Membranes

Abstract

Enzymatic digestion of membrane-bound proteins is one of the most widely used procedures for determining the internal amino acid sequence 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. Because the final step of protein purification is usually SDS-PAGE, electroblotting to either polyvinylidene difluoride (PVDF) or nitrocellulose is the simplest and most common procedure for recovering protein free of contaminants (e.g., SDS or acrylamide) with a high yield. As described in this unit, 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 membranes is higher than from nitrocellulose, particularly from higher-retention PVDF (e.g., ProBlott, Transblot, Westran, or Immobilon P(sp)). Finally, PVDF-bound protein can be stored dry, as opposed to nitrocellulose-bound protein, which must remain wet during handling and storage to prevent loss of peptides during digestion.