Protein and Peptide Recovery from PVDF Membranes

Abstract

Polyvinylidene difluoride (PVDF) is the most frequently utilized support for the sequence analysis of electroblotted proteins. This chapter describes a simple method for recovering intact proteins from PVDF membranes. A limitation of this technique is that N-terminal blocked proteins must be chemically cleaved or proteolytically digested to generate internal peptide fragments that can then be sequenced. The chapter describes in situ digestions on PVDF and nitrocellulose membranes. It presents the development of methods that directly extract protein from PVDF. These are based on solutions containing detergents that can limit subsequent proteolytic digestions or interfere with HPLC separations. A simple extraction procedure using the solvent dimethyl sulfoxide (DMSO) was developed that allows the recovery of intact proteins from PVDF membranes. The DMSO can be easily removed, allowing the extract to be analyzed by protein sequencing, amino acid analysis, or by other biochemical analysis. The extracted protein can be cleaved or digested and the resulting fragments can either be separated by HPLC or by tricine SDS-gels followed by a second round of electroblotting. An additional advantage of this technique is that the protein may be directly digested in the extraction solvent after addition of an appropriate buffer.