In Gel Slice Protease Digestion
1. Reduce and alkylate protein sample (see lab method). Run SDS-PAGE gel taking precautions against
modification (see lab method). Run as few lanes as possible to maximize concentration of
protein(s) within band(s) of interest. Run known amounts of molecular weight markers for the
purpose of estimating protein yield.
2. Stain gel with 0.1% Coomassie R250/20% MeOH/0.5% AcCOOH. Destain in 30% MeOH until the
bands are visible and the background is nearly clear.
3. Remove gel slices and trim away as much polyacrylamide as possible. Place gel slices in Eppendorf
brand 1.5 ml tube. Estimate volume of slices.
4. Add 1 ml of 0.05 M Tris-HCl, pH 9.0, 50% acetonitrile. Rotate gently for 1 hour at room
temperature. Remove wash and save.
· Prepare 100 mM Tris-HCl, pH 9.0. Dilute 1:1 with acetonitrile for this step. Use in step 7.
5. Repeat wash step. A third wash step may be used to reduce the amount of Coomassie in sample.
6. Dry completely in speed-vac.
7. To a volume of 100 mM Tris-HCl, pH 9.0 equal to the estimated volume of gel slices, add 0.1 - 0.2
µg of Endo Lys C (from Wako, Achromobacter lyticus). If amount of protein is known, add 1:10
molar ratio of protease to substrate (e.g., Endo Lys C is ~30 kDal. For 2 µg of a 30 kDal protein,
add 0.2 µg of Endo Lys C.)
8. Add Endo Lys C to dried gel slices. Add more 100 mM Tris-HCl, pH 9.0 as needed to rehydrate gel