Recently our lab has started protein and gel work. We have had
trouble digesting the proteins from an acrylamide gel matrix and
hope that some of you have suggestions. We are using apomyoglobin as
the trial protein, modified sequencing grade trypsin from Boehringer,
and 8% polyacrylamide gel under denaturing condition. We typically
stain with Coomassie Blue (0.1%) for 30min and destain with 40% MeOH,
10% acetic acid overnight with several changes of solution. After
destaining the bands are excised, washed several times with ammonium
bicarbonate and ACN to release the Coomassie stain. The gel pieces
are dried, disulfide bonds reduced with DTT, and treated with
iodoacetamide to prevent reformation of disulfide bonds. After
rinsing and drying the gel is swelled with the digestion buffer and
incubated overnight at 37C. Recovery is performed using 0.1%TFA and
ACN. We are trying to detect the peptides using a C18 narrow bore
column for which we have a method which works well with proteins
digested outside a gel. We think that we are losing the proteins at
one of these steps but have been unable to determine which so far.
Any suggestions would be greatly appreciated. Thanks,
Dan