Before you start
- For mass spectrometry, use colloidal G-250 and keratin-free handling (gloves, clean trays).
Reagents and materials
Totals are for 1 gels (+10% extra). Per-mini gel amounts are in the middle column.
| Reagent | Per mini gel | Total |
|---|---|---|
| Staining solution | 30 mL | 30 mL |
| Destaining solution | 100 mL | 100 mL |
Buffers and recipes
R-250 staining solution per 1 L
| Coomassie Brilliant Blue R-250 (0.1%) | 1 g |
| Methanol (40%) | 400 mL |
| Glacial acetic acid (10%) | 100 mL |
| Water | to 1 L |
Filter before use; reusable a few times.
Destaining solution per 1 L
| Methanol (40%) or ethanol | 400 mL |
| Glacial acetic acid (10%) | 100 mL |
| Water | 500 mL |
Equipment
- Staining tray
- Rocker
- Gel imager or scanner
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Fix and stain
Place the gel in staining solution and rock 30–60 min at room temperature.
Destain
Replace with destaining solution and rock, changing the solution until the background is clear (1–3 h or overnight). A folded tissue in the tray absorbs dye.
Image
Image on white light; store the gel in water or 5% acetic acid.
Critical points
- R-250 detects roughly 50–100 ng protein per band; colloidal G-250 detects around 10 ng.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Blue background | Destain longer with fresh solution. |
| Faint bands | Load more protein or use colloidal Coomassie or silver/fluorescent stains. |
Safety
Methanol and acetic acid: ventilated area, gloves.
Further reading
- Neuhoff V et al. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis (1988).
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.