Team:WashU/Protocols/Trypsin

Trypsin Digestion for LC/MS

1. Make, run, stain and destain SDS-PAGE gel as usual. Note: If protein is not being overproduced, purification may be necessary to give a clear band.
2. Excise protein band to be investigated using a razor blade. Following excision, mince this band into 0.5mm cubes and place in a low-bind eppendorf tube.
3. Destain gel pieces by adding 200ul of 200 mM ammonium bicarbonate and 40% acetonitrile and incubating for 30min at 37degrees
4. Repeat step 3 once more.
5. Dry gel pieces using a speed-vac for 30min.
6. Add 20 μl (0.4 μg of trypsin) of rehydrated trypsin to the tube.
7. Add 50 μl of 40 mM ammonium bicarbonate and 9% acetonitrile to the gel sample and mix.
8. Incubate mixture overnight.
9. The peptide pieces are now in the solution is now ready analyze by LC/MS. Simply remove the liquid, carefully avoiding the gel pieces.