In-gel tryptic digestion protocols
Using Promega Modified Sequencing Grade Trypsin (Porcine)
3 hour - overnight digestion
Wear gloves and a clean lab coat to avoid keratin contamination.
Prepare all solutions containing ammonium bicarbonate fresh daily.
Day 1
- Excise protein spot/band on a clean glass surface using a scalpel. Cut the gel as close to the band as possible to reduce the volume of gel to be processed. Cut into 4-5 pieces and place into a 500 µL microcentrifuge tube. Push gel pieces down with a pipet tip
- If storing the gel, add 40 L of 1% acetic acid in Milli-Q water. Store at 4 C until ready for digestion.
- Prepare the following solutions:
50mM Ammonium bicarbonate
0.0396 g in 10 mL of Milli-Q water
pH should be ~8
50mM Ammonium bicarbonate
0.0396 g in 10 mL of Milli-Q water
pH should be ~8
10 mM DTT in 50 mM ammonium bicarbonate
0.0015 g dithiothreitol, electrophoresis grade in 1 mL of 50 mM ammonium bicarbonate
100 mM iodoacetamide in 50 mM ammonium bicarbonate
0.0185 g iodoacetamide in 1 mL of 50 mM ammonium bicarbonate
50% Acetonitrile/25 mM ammonium bicarbonate
1 mL of acetonitrile
1 mL of 50 mM ammonium bicarbonate
- Remove the acetic acid from the tube.
- Destaining
Coomassie stained Bands. This can also be used to wash Sypro-Ruby stained gels
Wash the gel with 50 µL of 50 mM ammonium bicarbonate. Vortex mix and let stand for 5 minutes. Discard liquid into waste.
Shrink the gel with 50 µL of 50% acetonitrile/25 mM ammonium bicarbonate. Vortex mix and let stand for 10 minutes. Remove and discard the supernatant into waste.
Silver-stained bands
The silver-stained band/spot is incubated with a fresh mixture (v 1: 1) of 30 mM potassium ferricyanide (98 mg/10ml H2O) and 100mM sodium thiosulfate (248 mg/10ml H2O) for 15 minutes. Remove and discard the supernatant into waste.
Wash with 50 µL deionized water. Vortex mix and let stand for 5 minutes. Discard liquid into waste.
Wash the gel with 50 µL of 50 mM ammonium bicarbonate. Vortex mix and let stand for 5 minutes. Discard liquid into waste.
Shrink the gel with 50 µL of 50% acetonitrile/25 mM ammonium bicarbonate. Vortex mix and let stand for 10 minutes. Remove and discard the supernatant into waste.
- Reduction
Reduce with 30 µL of 10 mM DTT for 30 minutes at 56°C. Remove and discard the supernatant liquid into waste.
- Alkylation
Alkylate with 30 µL of 100 mM iodoacetamide for 15 minutes in the dark at room temperature. Remove and discard the supernatant liquid into waste.
- Shrink with 50 µL of 50% acetonitrile/25 mM ammonium bicarbonate. Vortex mix and let stand for 15 minutes. Remove and discard the supernatant into waste.
- Digestion
Prepare the trypsin solution:
Trypsin Stock (0.2 µg/µL): Add 100 µL of Promega resuspension buffer to 20 µg of lyophilized trypsin (porcine, modified sequencing grade). Store at -20ºC.
13 ng/µL trypsin in 50 mM ammonium bicarbonate
10 µL of trypsin stock
140 µL of 50 mM ammonium bicarbonate
Cover the gel pieces with 13 ng/ µL trypsin to the gel pieces. Incubate for at least 3 hours at 37°C.
- Extraction of Peptides
Prepare the following solutions:
25 mM ammonium bicarbonate
1 mL of 50mM ammonium bicarbonate
1 mL of Milli-Q water
5% formic acid in water
4.75 mL of Milli-Q water
250 µL of formic acid
Remove the tubes from the incubation. From this point on the supernatant will be collected into a new 500 µL microcentrifuge tube.
Transfer the liquid to a new tube.
Add 20 µL of 25 mM ammonium bicarbonate. Vortex mix and let stand for 10 minutes. Add to the supernatant collected in step b).
Add 20 µL of 5% formic acid. Vortex mix and let stand for 10 minutes. Add to the supernatant collected in step b).
Add 20 µL of 100% acetonitrile. Vortex mix and let stand for 10 minutes. Add to the supernatant collected in step b).
Add 20 µL of 5% formic acid. Vortex mix and let stand for 10 minutes. Add to the supernatant collected in step b).
Add 20 µL of 100% acetonitrile. Vortex mix and let stand for 10 minutes. Add to the supernatant collected in step b).
- Store the extracted peptides at -20°C until ready to evaporate down.
- Preparation of Analysis
Evaporate down to dryness and reconstitute in 5 µL of 0.1% formic acid in water or 0.1% TFA in water. Samples are ready for LC MS/MS analysis with no further clean-up needed. Samples will need to undergo C18 Ziptip cleanup before ESI or MALDI MS analysis.