In-gel digestion has become a standard procedure for mass spectrometry-based protein identification. The procedure takes advantage of the incredible sensitivity of the mass spectrometer by producing peptides by protease digestion within the acrylamide band or spot. The resulting peptides are extracted and analyzed by the mass spectrometer followed by computer-based database searching for matches to the peptide masses.
Our recommendations:
Use Coomassie stained bands or spots. Mass spec-compatible silver staining procedures exist (see our Protocols page) but often the amount of protein detected by silver is too small for successful analysis. Consult this link to try to estimate 50 pmol of Coomassie stained protein.
Silver stained bands (or spots) should be de-stained prior to digestion (see our Protocols page). Your silver staining protocol MUST be mass spectrometry compatible. Please consult with the PCL staff before bringing samples stained (or destained) with silver. We discourage bringing silver stained bands (or spots) for Peptide Mass Fingerprinting or LC/MS/MS. SILVER STAINING WILL REDUCE THE LIKELIHOOD OF USEABLE DATA.
Fluorescent stains (Sypro, Cy) are acceptable, but like silver, the stains are very sensitive and the amount of protein detected may be too small for successful analysis. Consult with the PCL staff before bringing the samples.
Cut the band (or spot) from a single lane. We recommend a 0.75 mm thick gel slice. The rule of thumb is to keep the ratio of protein:acrylamide as high as possible. Acrylamide is an interfering agent in the analysis. Trim the band (or spot) to eliminate ’empty’ acrylamide. We prefer that you bring the uncut gel to the PCL. Let us cut the bands.
Reduce and alkylate the protein (in the gel slice if necessary) before digestion (see our Protocols page). Our experience is that this will increase the number and amount of peptides obtained.
WEAR GLOVES DURING ALL PROCEDURES AND USE THE HIGHEST PURITY REAGENTS AND SOLVENTS AVAILABLE.