We do our in-gel Endoproteinase GluC digests in the same manner as Trypsin in-gel digests, using our GluC digestion buffer to re-swell the gel slices after they are dehydrated using acetonitrile and adding Endoproteinase GluC for a 1:10 or 1:20 wt/wt protein:GluC ratio based on an estimation of the amount of protein present in the gel band. For example, to 1 ug protein add 50 to 100 ng of Endoproteinase GluC and incubate at 25- 37°C overnight. Using the GluC digestion buffer provided which contains Glu-Glu dipeptide to re-swell the gel slice is key to achieving good digestions with Endoproteinase GluC. Unlike Trypsin digests, Endoproteinase GluC digestions do not reach a reaction endpoint due to the 100-300 fold difference in cleavage rates between Glu and Asp residues: when all of the Glu residues have been cleaved, the enzyme is still digesting Asp residues. For reproducibility, we suggest all Endoproteinase GluC reactions be for the same duration. This is in contrast to Trypsin digests, which go to completion and thus can have flexible incubation times. We generally clean-up our in-gel reactions on C18 ZipTips before MS (MALDI-MS or Ion Trap MS/MS).