Before you start
- Pre-cool the centrifuge and keep everything on ice.
- Add inhibitors to RIPA just before use.
Reagents and materials
Totals are for 6 samples (+10% extra). Per-sample (6-well or 10⁶ cells) amounts are in the middle column.
| Reagent | Per sample (6-well or 10⁶ cells) | Total |
|---|---|---|
| Ice-cold PBS (wash) | 2 mL | 2 mL |
| RIPA buffer + inhibitors | 150 µL | 150 µL |
| Protease inhibitor cocktail (100×) | 1.5 µL | 1.5 µL |
| Phosphatase inhibitor cocktail (100×) | 1.5 µL | 1.5 µL |
Buffers and recipes
RIPA lysis buffer per 100 mL
| 1 M Tris-HCl, pH 7.4 (50 mM) | 5 mL |
| 5 M NaCl (150 mM) | 3 mL |
| NP-40 or Triton X-100 (1%) | 1 mL |
| 10% sodium deoxycholate (0.5%) | 5 mL |
| 10% SDS (0.1%) | 1 mL |
| 0.5 M EDTA (1 mM) | 0.2 mL |
| Ultrapure water | to 100 mL |
Store at 4 °C. Add protease and phosphatase inhibitor cocktails just before use.
Equipment
- Cell scraper
- Microcentrifuge at 4 °C
- Sonicator (optional)
- Ice
Procedure
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Wash cells
Place the plate on ice, aspirate medium and wash twice with ice-cold PBS.
Lyse
Add 100–150 µL RIPA (with inhibitors) per well, scrape and collect into a cold tube. For tissue, homogenise in 10 volumes of buffer.
Incubate
Keep 30 min on ice, vortexing briefly every 10 min.
Shear DNA (optional)
Sonicate briefly (3 × 5 s pulses on ice) to reduce viscosity.
Clear
Centrifuge at 14,000–16,000 × g for 15 min at 4 °C.
Collect
Transfer the supernatant to a new tube; keep an aliquot for protein assay.
Store
Use immediately or store aliquots at −80 °C; avoid repeated freeze–thaw.
Critical points
- Phosphatase inhibitors are essential for phospho-protein work.
- RIPA does not fully solubilise some nuclear or membrane proteins — consider SDS lysis buffer.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Viscous lysate | Sonicate or add nuclease. |
| Degraded bands | Work cold and fast; fresh inhibitors. |
| Low yield | Use more cells, less buffer, or a stronger buffer. |
Safety
SDS and detergents are irritants.
Further reading
- Abcam / CST technical guides on sample preparation for western blotting.
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.