Western blot stripping buffer (harsh, β-ME) recipe
For antibodies that survive mild stripping: 62.5 mM Tris-HCl pH 6.8, 2% SDS, 100 mM β-mercaptoethanol at 50 °C.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| 1 M Tris-HCl (pH 6.8) | 6.25 mL | 62.5 mM |
| 10% SDS | 20 mL | 2% |
| β-mercaptoethanol (add fresh) | 700 µL | 100 mM |
| Water (distilled) | to final volume |
Steps
- Mix Tris and SDS with water; add β-ME in a fume hood just before use.
- Incubate the membrane at 50 °C for 30 min with agitation.
- Wash thoroughly with TBST (5–6 × 5 min) to remove β-ME.
- Re-block and probe with the next primary antibody.
Good to know
- Check with ECL alone that the old signal is gone before re-probing.
- Some antigen is also lost, so do not compare band intensities across stripping.
⚠ Safety β-ME is toxic and malodorous — handle and heat it in a fume hood.
FAQ
Mild or harsh first?
Try mild first and use harsh only if needed. PVDF tolerates it better than nitrocellulose.
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.