Representative "In-Gel" Digestion Protocol for Proteins in SDS PAGE Gel
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% AcOH, 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 digested with trypsin. A reduction and alkylation step is included. Alternatively, the sample may be reduced and alkylated prior to electrophoresis. Note that an alternate buffer systemis also provided for LysC digestion.
- Wash the gel slices for at least 1 hr in 500 microliters of 100 mM ammonium bicarbonate. Discard the wash.
- Add 150 microliters of 100 mM ammonium bicarbonate and 10 microliters of 45 mM DTT. Incubate at 60 degrees centigrade for 30 min.
- Cool to room temp and add 10 microliters of 100 mM iodoacetamide and incubate for 30 min in the dark at room temperature.
- Discard the solvent and wash the gel slice in 500 microliters of 50% acetonitrile/100 mM ammonium bicarbonate with shaking for 1 hr. Discardthe wash. Cut the gel into 2-3 pieces and transfer to a 200 microliter eppendorf style PCR tube.
- Add 50 microliters of acetonitrile to shrink the gel pieces. After 10-15 min remove the solvent and dry the gel slices in a rotatory evaporator.
- Re-swell the gel pieces with 10 microliters of 25 mM ammonium bicarbonate containing Promega modified trypsin (sequencing grade) at a concentration such that a substrate to enzyme ratio of 10:1 has been achieved. (If the amount of protein is not known, add 0.1-0.2 microgramsof modified trypsin in 10 microliters of 25 mM ammonium bicarbonate).After 10-15 minutes add 10-20 microliters of additional buffer to cover the gel pieces. Gel pieces need to stay wet during the digest. Incubate 4 hrs to overnight at 37 degrees Centigrade.
Proceed to step 8 if further extraction of the gel is desired (recommended)- otherwise continue with step 7.
- Approximately 0.5 microliters of the supernatant may be removed for MALDI analysis and/or the supernatant acidified by adding 10% TFA to a final concentration of 1% TFA for injection onto a narrow- or microbore reverse phase column. (If necessary the sample's volume may be reduced~1/3 on a rotatory evaporator.)
- Extraction (Optional)- Save supernatant from step 7 in tube X, and extract peptides from gel twice with 50 microliters of 60%acetonitrile/0.1% TFA for 20 min. Combine all extracts in tube X (using the same pipet tip to minimize losses), and speed vac to near dryness.Reconstitute in 20 microliters of appropriate solvent. Proceed with chromatography or MALDI analysis.
Alternate Digestion Protocol.
If a LysC digest is desired a different buffer system is used, using the same volumes as before. This buffer system (below) has been used successfully with LysC from Achromobacter lyticus. LysC from other sources has not been tested.
- Wash the gel pieces in 500 mM TrisHCl pH 9.2/50% acetonitrile.
- Digest the protein(s) in the gel slice with LysC in 100 mM TrisHCl pH 9.2.