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"In-Gel" Trypsin Digestion Protocol for Proteins in SDS-PAGE Gel Slices ABRF Internal Protein Sequence Research Committee (11/97)
 

Summary: 98
"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 l of 100 mM ammonium bicarbonate. Discard wash.
2. Add 150 l of 100 mM ammonium bicarbonate and 10 l of 45 mM DTT. Incubate at 60C for 30
min.
3. Cool to room temp and add 10 l of 100 mM iodoacetamide and incubate for 30 min in the dark at
room temperature.
4. Discard the solvent and wash the gel slice in 500 l 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 l eppendorf style PCR tube.
5. Add 50 l 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 l of 25 mM ammonium bicarbonate containing Promega modified

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine