If there is a choice, the lower the percentage of acrylamide, the better
the in-gel digestion will work. Also, its best to let the running gel sit a
bit before running the samples (say 2 hrs or more), because residual
peroxides can cross-link the protein to the gel and reduce extraction of
the peptides. Peroxides in the stacking gel don't matter, so the stacker
can be freshly made. We get the best results from in-gel digests if the
gels are run fairly fast or if we set the running gels sit overnight before
using them.
We stain in 0.5% R250 Coomassie, 25% isopropanol, 10% acetic, destain in
10% acetic. After cutting out the bands, we destain in 50% acetonitrile
several times. A little blue doesn't hurt. Desalting before the maldi is
really essential, unless you have a lot of protein.
Katheryn Resing