PVDF Membrane Digestion

This protocol is used in our laboratory on Coomassie or Amido Black stained proteins on PVDF membranes. We routinely use Promega modified trypsin or Endo Lysine C (EKC) from Waco Chemical Co. EKC cleaves after Lysines only and produces larger fragments. Keep in mind that larger fragments may be more hydrophobic and not extract well from the PVDF membrane. We recommend starting with a minimum of 100 pmol of protein for peptides destined to automated Edman sequencing. Less material can be used for mass spec analysis and subsequent protein identification. Although this is still a popular method, it has become superceded by the protocol for digesting proteins in gel slices. For this protocol, as all others, wear gloves and always use the highest quality reagents and chemicals. This is a modification of a protocol shared with us by Steve Tindall, Argo BioAnalytica, Inc, Morris Plains, New Jersey.