UNIT 11.2 Enzymatic Digestion of Proteins on PVDF Membranes
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
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.
- Published Online: 1 MAY 2001
- Published Print: JUN 1995
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.