Bradford protein assay reagent (homemade) recipe
A protein assay reagent based on Coomassie G-250 shifting to 595 nm absorbance when bound to protein: 100 mg Coomassie G-250, 50 mL 95% ethanol, 100 mL 85% phosphoric acid per litre.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| Coomassie Brilliant Blue G-250 | 50 mg | 0.01% |
| 95% ethanol | 25 mL | 4.75% |
| 85% phosphoric acid (H₃PO₄) | 50 mL | 8.5% |
| Water (distilled) | to final volume |
Steps
- Dissolve the Coomassie G-250 in the ethanol.
- Add the phosphoric acid and dilute to volume with water.
- Filter through Whatman No. 1 paper and store in a brown bottle at room temperature (weeks).
Good to know
- Mix 5–10 µL sample with 200–250 µL reagent (96-well), read 595 nm after 5 min and compare with a BSA standard curve.
- Detergents such as SDS and Triton interfere — use BCA for RIPA lysates.
⚠ Safety Concentrated phosphoric acid is corrosive — wear gloves and goggles.
FAQ
My standard curve is not linear.
Bradford is inherently non-linear over wide ranges. Use a narrow range or a quadratic fit — the standard-curve tool picks one automatically.
This is a widely used standard composition. Amounts depend on the hydrate form and purity of your reagents — check the formula weight (FW) on the bottle and follow your institution's safety rules and the manufacturers' instructions.