Cytoplasmic extraction buffer (hypotonic, buffer A) recipe
The hypotonic buffer for the first step of nuclear/cytoplasmic fractionation — cells swell and only the plasma membrane breaks: 10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| 1 M HEPES-KOH (pH 7.9) | 500 µL | 10 mM |
| 1 M KCl | 500 µL | 10 mM |
| 1 M MgCl₂ | 75 µL | 1.5 mM |
| 1 M DTT (add fresh) | 25 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.
- Swell cells on ice for 10–15 min, add NP-40 to 0.3–0.6% and vortex to break the plasma membrane.
- Centrifuge briefly (a few thousand g, 1 min): the supernatant is cytoplasm, the pellet contains nuclei.
Good to know
- Check fractionation by western blot with cytoplasmic (GAPDH, tubulin) and nuclear (lamin B1, histone H3) markers.
FAQ
Can I skip the NP-40?
You can break swollen cells with a narrow needle or Dounce homogeniser instead; check lysis under the microscope.
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.