RIPA Lysis Buffer Recipe: Composition, Preparation and When to Use It

TL;DR: A common RIPA formulation contains 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% non-ionic detergent, 1% sodium deoxycholate and 0.1% SDS. Add protease/phosphatase inhibitors fresh when required. RIPA is strong enough for broad protein extraction but can disrupt native protein complexes.

RIPA buffer is a high-stringency lysis buffer used for extraction of cytoplasmic, membrane and many nuclear proteins before Western blotting and related protein assays. Its combination of non-ionic and ionic detergents makes it more disruptive than NP-40-only buffers.

Standard RIPA composition used in this guide

100 mL RIPA recipe from common stocks

• 2.5 mL 1 M Tris-HCl, pH 7.6
• 3.0 mL 5 M NaCl
• 10 mL 10% non-ionic detergent stock
• 10 mL 10% sodium deoxycholate
• 1 mL 10% SDS
• Purified water to 100 mL

Keep a base RIPA stock without unstable inhibitors. Add protease inhibitors and, where phosphorylation must be preserved, phosphatase inhibitors immediately before lysis according to the inhibitor system being used.

Preparation

1. Combine Tris-HCl and NaCl in approximately 60–70 mL purified water.
2. Add the non-ionic detergent slowly to minimise foaming.
3. Add sodium deoxycholate and SDS and mix until homogeneous.
4. Bring to 100 mL with purified water.
5. Store the inhibitor-free stock cold in a clean bottle.
6. Add fresh inhibitors only to the aliquot needed for the experiment.

How much RIPA buffer should I use?

The minimum useful volume depends on cell number, tissue mass and viscosity. For cultured cells, use enough buffer to fully wet and suspend the pellet or scraped monolayer without unnecessarily diluting the protein. For tissue, mechanical homogenisation is often required.

RIPA extraction workflow

1. Keep samples and buffer cold.
2. Add freshly supplemented RIPA to the sample.
3. Mix thoroughly and incubate on ice for approximately 10–30 minutes.
4. Shear viscous genomic DNA by brief sonication or repeated passage through a narrow-gauge needle if compatible with the target.
5. Clarify the lysate by high-speed centrifugation at 4°C.
6. Transfer the supernatant to a clean tube and quantify protein using a detergent-compatible assay.

When RIPA is a good choice

• Whole-cell lysates for Western blotting
• Extraction of many membrane-associated proteins
• Samples where high solubilisation is more important than preserving native complexes
• Workflows using detergent-compatible protein assays

When RIPA is the wrong choice

RIPA can disrupt protein-protein interactions and partially denature proteins. For co-immunoprecipitation, pull-down assays or native enzyme assays, start with a milder non-ionic lysis buffer unless the interaction has already been shown to survive RIPA conditions.

RIPA vs NP-40 buffer

NP-40-style buffers are milder because they typically omit SDS and deoxycholate. RIPA therefore extracts a broader protein population but is more likely to disrupt native complexes. Published comparisons also show that extraction yield and protein coverage depend on tissue type and downstream analytical method.

Troubleshooting

Frequently asked questions

What does RIPA stand for?

Radioimmunoprecipitation assay buffer. The name persists even though RIPA is now widely used simply as a protein extraction buffer.

Can RIPA be used for mass spectrometry?

Detergents can interfere with LC-MS, so a cleanup strategy is required. Primary studies show modern sample-preparation workflows can accommodate RIPA, but compatibility should be validated for the specific workflow.

Related ABMIUM guides

• Western Blot Protocol
• Co-IP Protocol
• Pull-Down Assay Protocol

Primary research references

1. Elinger D, Gabashvili A, Levin Y. S-Trap compatibility with common protein extraction buffers and detergents. J Proteome Res. 2019.
2. Sample preparation for breast-cancer proteomics: comparison of RIPA and urea lysis buffers.
3. Peach M, Marsh N, Macphee DJ. Protein solubilization and lysis-buffer choice. Methods Mol Biol. 2012.