"In-Gel" Trypsin Digestion Protocol for Proteins in SDS-PAGE Gel Slices
ABRF Internal Protein Sequence Research Committee (11/97)
Samples to be digested in the gel are run in as few lanes as possible to maximize the concentration of the
protein within the bands of interest. The gel is stained in 0.1% Coomassie R250/20% MeOH/0.5%
AcCOOH, and then destained in 30% MeOH until the bands are visible and the background is nearly clear.
Volumes and reagent quantities described herein are for one band from a 1 mm gel slice. A reduction
and alkylation step is included. Alternatively, the sample may be reduced and alkylated prior to
electrophoresis. Note that an alternate buffer system is also provided for LysC digestion.
1. Wash the gel slices for at least 1 hr in 500 µl of 100 mM ammonium bicarbonate. Discard wash.
2. Add 150 µl of 100 mM ammonium bicarbonate and 10 µl of 45 mM DTT. Incubate at 60°C for 30
3. Cool to room temp and add 10 µl of 100 mM iodoacetamide and incubate for 30 min in the dark at
4. Discard the solvent and wash the gel slice in 500 µl of 50% acetonitrile/100 mM ammonium
bicarbonate with shaking for 1 hr. Discard the wash. Cut the gel into 2-3 pieces and transfer to a
200 µl eppendorf style PCR tube.
5. Add 50 µl of acetonitrile to shrink the gel pieces. After 10-15 min remove the solvent and dry the
gel slices in a rotatory evaporator.
6. Re-swell the gel pieces with 10 µl of 25 mM ammonium bicarbonate containing Promega modified