I believe that the concentration of methanol and acetic acid used during
staining is critical for obtaining good in-gel digestion with a good yield of
peptide fragments. Not only is the concentration of these components important
in determining whether a protein is fixed in the gel but the amount of time
spent in these solutions is also critical.
You have described a standard procedure for Coomassie Blue staining of proteins
in SDS-PAGE analysis. However, you did not specify how long the gel was stained
or how long the gel was destained under the conditions described. I generally
use 0.05% Coomassie Brilliant Blue-G in 10% MeOH, 5% acetic acid for staining.
Stain for up to 1 hour, and then destain in 10% MeOH, 5% Acetic acid only until
the band of interest is visible and can be excised. No need to destain
completely, because the subsequent steps (washes, in-gel reduction and
alkylation, washes etc.) will remove any excess dye. I would never store the
gel in 10% acetic acid prior to in-gel digestion, nor have I ever seen this in
any protocol. If more sensitive staining techniques are required there are
numerous other possibilities that work extremely well for in-gel digestion
(modified silver stain, zinc reverse stain, and fluorescent stains).
Hope this helps.
Raymond Boynton
Structural Biochemistry
Biogen, Inc.