Lysis buffers for western blot and immuno­precipitation: RIPA, NP-40 and native buffers

A lysis buffer breaks open cells and releases their proteins in a form your next step can use. A western blot only needs the proteins solubilised, because they will be denatured anyway. An immunoprecipitation needs the target folded well enough for an antibody to recognise it, and a co-IP needs its binding partners still attached. An enzyme assay needs the protein active.

Those goals pull in different directions, which is why there is no single best lysis buffer. The detergent is the main dial: ionic detergents such as SDS and sodium deoxycholate extract strongly and denature; non-ionic detergents such as NP-40 and Triton X-100 extract more gently.

The original Nonidet P-40 is no longer manufactured; IGEPAL CA-630 is the chemically equivalent replacement sold under that name in most recipes.

• Protease inhibitors: a broad cocktail, or PMSF at about 1 mM with aprotinin and leupeptin. Add them just before use; PMSF breaks down within an hour in water.
• Phosphatase inhibitors: for phospho-proteins, sodium orthovanadate (about 1 mM, activated), sodium fluoride (about 10 mM) and β-glycerophosphate.
• EDTA: inhibits metalloproteases but also strips metal from metal-dependent enzymes and His-tagged proteins, so leave it out for those.
• Nuclease or sonication: breaks up released DNA, which otherwise makes RIPA lysates viscous and hard to pipette.

1. Place the dish on ice and wash cells twice with ice-cold PBS.
2. Add cold lysis buffer with inhibitors, about 0.5–1 mL for a 100 mm dish, and scrape the cells into a cold tube.
3. Incubate on ice for 15–30 minutes, mixing occasionally.
4. Centrifuge at 12,000–14,000 × g for 15 minutes at 4 °C.
5. Transfer the supernatant to a fresh cold tube. This is your lysate.
6. Measure protein concentration, then use immediately or store at -80 °C in aliquots.

For western blot, mix with Laemmli sample buffer and heat at 95 °C for 5 minutes. Some multi-pass membrane proteins aggregate when boiled; heat them at 37–70 °C instead.

Red blood cell (RBC) lysis buffer is not for extracting proteins. It is an ammonium chloride solution that bursts red cells while leaving white cells intact, used to prepare leukocytes for flow cytometry or for DNA and RNA extraction from blood. It is often sold as a 10X stock to dilute to 1X before use. For nucleic acids from blood, see the extraction by sample type guide.

Once you have a clean lysate, the antibody is the next variable. The antibody range lists validated applications for each antibody, and the applications guide covers the controls for western blot and IP. To quantify a protein instead of visualising it, use an ELISA kit on the same lysate.