RIPA buffer is one of the most common lysis buffers in protein research, and making it from stock solutions takes about 10 minutes. The standard recipe combines five components in water: Tris-HCl for pH buffering, NaCl for ionic strength, and three detergents (NP-40, sodium deoxycholate, and SDS) that work together to break open cells and solubilize proteins. Here’s how to prepare 100 mL from scratch.
The Standard RIPA Recipe
Final working concentrations for RIPA buffer are consistent across most published protocols:
- Tris-HCl, pH 7.4–8.0: 25 mM
- NaCl: 150 mM
- NP-40 (or Triton X-100): 1%
- Sodium deoxycholate: 1%
- SDS: 0.1%
You’ll see minor variations between sources. Some older protocols use 10 mM Tris instead of 25 mM, and pH targets range from 7.4 to 8.0. Either end of that range works well for most applications. The 25 mM Tris, pH 7.6 formulation from Thermo Fisher has become the most widely referenced version.
Making 100 mL From Stock Solutions
Most labs prepare RIPA from concentrated stock solutions rather than weighing out dry chemicals each time. Here’s a practical recipe for 100 mL, adapted from a commonly used protocol:
- 1 M Tris-HCl (pH 7.4): 2.5 mL
- 5 M NaCl: 3.0 mL
- 20% NP-40: 5.0 mL
- 10% sodium deoxycholate: 10.0 mL
- 20% SDS: 0.5 mL
- Distilled water: bring to 100 mL
If your lab no longer stocks NP-40 (it’s been discontinued by some suppliers), Triton X-100 is a widely accepted substitute at the same concentration. The two detergents behave almost identically for cell lysis.
Add the Tris-HCl first, then the NaCl, followed by the detergents. Save the SDS for last, since it can precipitate if it contacts high concentrations of salt before being diluted. Top up with distilled water and mix gently. There’s no need to adjust the pH separately if your Tris stock is already at the correct pH.
Adding Protease and Phosphatase Inhibitors
The base RIPA buffer breaks open cells, but it won’t stop enzymes from degrading your proteins once they’re released. Protease inhibitors and phosphatase inhibitors need to be added fresh, right before you use the buffer. They lose activity quickly once diluted, so never add them to your entire stock bottle.
The most common additions are PMSF (a serine protease inhibitor, typically at 1 mM), EDTA (a metalloprotease inhibitor, 1–5 mM), and sodium orthovanadate (a phosphatase inhibitor, 1 mM). Many researchers now use commercial protease inhibitor cocktails, which are sold as concentrated tablets or liquid mixes that you add to a set volume of buffer. These cocktails cover a broader range of proteases than PMSF alone.
If you’re studying phosphorylated proteins, phosphatase inhibitors are essential. If you only need total protein levels and don’t care about phosphorylation status, you can skip them.
How Much Buffer to Use Per Sample
A good starting point for cultured cells is about 100–200 µL of RIPA per million cells, or roughly 1 mL per 10 cm dish of confluent cells. For tissue samples, 1 mL per 50–100 mg of tissue is typical. Using too little buffer means you won’t fully lyse your sample. Using too much dilutes your protein concentration, which can cause problems downstream when you’re loading gels or running assays.
After adding buffer, keep the lysate on ice for 15–30 minutes with occasional vortexing, then centrifuge at high speed (around 14,000 × g for 15 minutes at 4°C) to pellet the insoluble debris. The supernatant is your protein lysate.
Storage and Shelf Life
RIPA buffer without inhibitors is stable at 4°C for several months. Store it in the refrigerator rather than at room temperature, but be aware that SDS can precipitate out of solution in the cold. If you see white crystals or cloudiness when you pull the bottle out, warm it briefly to room temperature and mix until the solution clears. The buffer is still fine to use.
For long-term storage, some labs keep aliquots at -20°C. This works, but you’ll almost certainly see SDS precipitation after thawing, so plan on warming and mixing each aliquot before use. Never freeze buffer that already contains protease inhibitors, since most of them degrade through freeze-thaw cycles.
When Standard RIPA Isn’t Enough
RIPA is considered a moderately harsh lysis buffer. The combination of three detergents solubilizes most cytoplasmic and membrane-associated proteins while still preserving many protein-protein interactions. That makes it a solid default choice for western blots.
There are situations where you might need to adjust the recipe. If you’re working with tightly bound membrane proteins that aren’t coming into solution, increasing the SDS concentration to 0.5% or even 1% can help, though this will disrupt most protein complexes. On the other hand, if you’re doing co-immunoprecipitation and need to keep protein interactions intact, consider dropping the SDS entirely and reducing sodium deoxycholate to 0.25–0.5%. Some researchers switch to a milder NP-40-only buffer for these experiments.
Nuclear proteins can also be tricky. RIPA generally lyses both the outer cell membrane and the nuclear envelope, but very tightly chromatin-bound proteins may require sonication or the addition of a nuclease to release them into solution. If your target protein is nuclear and you’re getting poor yields, brief sonication of the lysate (a few short pulses on ice) often solves the problem.
Common Mistakes to Avoid
The most frequent issue is forgetting to add inhibitors fresh. If you added PMSF to your stock bottle last week, it’s long since degraded. PMSF has a half-life of about 30 minutes in aqueous solution, so it needs to go in immediately before lysis.
Another common problem is letting samples warm up. All lysis steps should happen on ice or at 4°C. Proteases become much more active at room temperature, and even a few minutes of warmth can degrade sensitive targets. Pre-chill your RIPA buffer before adding it to cells or tissue.
Finally, skipping the centrifugation step leaves lipids and cellular debris in your lysate, which can cause smearing on gels and inconsistent protein quantification. Always spin down your lysate and transfer only the clear supernatant to a fresh tube.