Nuclear extraction buffer (high-salt, buffer C) recipe
Extracts nuclear proteins from the nuclear pellet with high salt: 20 mM HEPES pH 7.9, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 25% glycerol.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| 1 M HEPES-KOH (pH 7.9) | 200 µL | 20 mM |
| 5 M NaCl | 840 µL | 420 mM |
| 1 M MgCl₂ | 15 µL | 1.5 mM |
| 0.5 M EDTA | 4 µL | 0.2 mM |
| Glycerol | 2.5 mL | 25% |
| 1 M DTT (add fresh) | 5 µL | 0.5 mM |
| Water (distilled) | to final volume |
Steps
- Mix everything except DTT with water and store at 4 °C.
- Add DTT and protease inhibitors just before use.
- Resuspend the nuclear pellet and keep on ice for 30 min with occasional vortexing.
- Spin at maximum speed for 10–15 min; the supernatant is the nuclear extract.
FAQ
Is it suitable for EMSA?
Yes — this Dignam-type method was designed for transcription-factor binding assays such as EMSA; dialyse to lower the salt if needed.
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.