In Gel Digestion

This procedure is used routinely in our laboratory to prepare peptides for protein sequence analysis and protein identification procedures using mass spectrometry. The method is designed for Coomassie stained proteins and is successful, in our hands, in more than 90% of experiments. We recommend that you start with a minimum of 50 pmol of stained protein for peptides destined for Edman sequence analysis and >10 pmol of protein for mass spec analysis. Clearly, the more the better. We further recommend that you have the protein in a single gel slice to reduce the amount of acrylamide present. Your goal is to obtain a high protein/acrylamide ratio for maximal peptide retrieval. This procedure can also be applied to proteins that are stained with silver IF a non-fixing method was employed. We routinely use a method published by Blum (see references and Gel Staining) which is reversible. The protocol to destain (Blum) silver-stained gels is included at the bottom of this page. In our hands, destaining silver stained proteins can double the amount of peptides retrieved from digestion.

Reduction and alkylation is considered optional. Many labs omit these steps from their procedures. Our experience has been that enough peptides are retrieved for mass spec or Edman sequence analysis if it is omitted and, often, the additional steps actual reduce recoveries and increase contamination. For this protocol, as well as all others, use the highest quality reagents and chemicals available. Wear powder-free gloves as keratins from your skin are common contaminants in these experiments.

We routinely use Promega modified trypsin for digests but also recommend Endo Lys C (EKC) (Waco Chemical Co). EKC cleaves only after Lysines and, therefore, produces larger peptides than trypsin.
This is a modification of a method published by Williams and Stone (see references).