I get 5 bands of my protease enzyme out of which I have to purify the band which is responsible for protease activity and then I want to go for its identification.
Just go for a normal tryptic analysis of the cut out gel spots...
check out this paper for the procedure
Determination of site-specific glycan heterogeneity on glycoproteins.
Kolarich D, Jensen PH, Altmann F, Packer NH.
Nat Protoc. 2012 Jun 7;7(7):1285-98. doi: 10.1038/nprot.2012.062.
PMID: 22678432
U can add particular inhibitor, and run gel. Which band is missing find out........i think it will work.
My simple way to solve this problem was just to cut out unstained bands (run two lines, stain one, keep the other in buffer meanwhile), crush the gel (I just passed it through syringe several time), extract overnight at 4°C and measure activity as usually. After this I have easily identified my protein.
You can try copper staining the gel, this is a reverse stain and so the bands show up against a green/ blue opaque background. The advantage of this satin is it doesn't interfere with many protein identification techniques e.g. MALDI. The gel pieces can be stored for further analysis or crushed and extracted.
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