Unit

UNIT 11.2 Enzymatic Digestion of Proteins on PVDF Membranes

  1. Joseph Fernandez,
  2. Sheenah M. Mische

Published Online: 1 MAY 2001

DOI: 10.1002/0471140864.ps1102s00

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Fernandez, J. and Mische, S. M. 2001. Enzymatic Digestion of Proteins on PVDF Membranes. Current Protocols in Protein Science. 00:11.2:11.2.1–11.2.10.

Author Information

  1. The Rockefeller University, New York, New York

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: JUN 1995

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 Psp). 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.