Before you start
- Cut bands on a clean glass plate wearing gloves; include a blank gel piece as control.
Reagents and materials
Totals are for 6 bands (+10% extra). Per-gel band amounts are in the middle column.
| Reagent | Per gel band | Total |
|---|---|---|
| Destain: 50 mM ABC / 50% acetonitrile | 1 mL | 1 mL |
| Acetonitrile (dehydration) | 0.5 mL | 0.5 mL |
| 10 mM DTT in 50 mM ABC | 100 µL | 100 µL |
| 55 mM iodoacetamide in 50 mM ABC (fresh, dark) | 100 µL | 100 µL |
| Trypsin, 10–20 ng/µL in 50 mM ABC | 30 µL | 30 µL |
| Extraction: 50% acetonitrile / 5% formic acid | 200 µL | 200 µL |
Buffers and recipes
50 mM ammonium bicarbonate (ABC) per 100 mL
| NH₄HCO₃ | 0.395 g |
| LC-MS-grade water | to 100 mL |
Make fresh (pH ≈ 7.8–8.0; do not adjust).
Equipment
- Clean scalpel
- Low-binding tubes
- Thermomixer
- Vacuum concentrator
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Excise and dice
Cut the band and dice into ~1 mm cubes.
Destain
Wash with 50 mM ABC / 50% acetonitrile, 15–30 min with shaking, repeating until clear.
Dehydrate
Add acetonitrile until pieces turn white and shrink; remove.
Reduce
Add 10 mM DTT and incubate 30–45 min at 56 °C.
Alkylate
Replace with 55 mM iodoacetamide and incubate 30 min at room temperature in the dark.
Wash and dehydrate
Wash with ABC, dehydrate with acetonitrile and dry briefly.
Add trypsin
Cover with cold trypsin solution and keep on ice 30 min to swell; add ABC to cover.
Digest
Incubate overnight at 37 °C.
Extract peptides
Collect the solution, then extract twice with 50% acetonitrile / 5% formic acid (15 min each) and pool.
Dry
Dry in a vacuum concentrator and desalt or resuspend for LC-MS/MS.
Critical points
- Keratin from skin and dust is the main contaminant: clean tools, gloves, covered tubes.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Low identification | Use more protein, check destaining and extraction, avoid over-drying gel pieces before trypsin. |
Safety
Acetonitrile is flammable and toxic; iodoacetamide is toxic.
Further reading
- Shevchenko A et al. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature Protocols (2006).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.