Making a lysis buffer means combining a detergent to break open cells, a salt to control ionic strength, a pH buffer, and protective additives to keep your target molecules intact. The exact recipe depends on what you’re extracting and what you plan to do with it afterward. Below are the most common formulations, what each component does, and the practical steps to get consistent results.
Choose Your Buffer Based on Your Application
The single biggest decision is whether you need a denaturing or non-denaturing buffer. Denaturing buffers (like RIPA) unfold proteins and break apart complexes, which is ideal for Western blots where you’ll separate proteins by size anyway. Non-denaturing buffers keep proteins in their native shape, which matters for co-immunoprecipitation, enzyme activity assays, or any experiment where protein-protein interactions need to stay intact.
If you’re unsure, RIPA buffer is the most widely used general-purpose lysis buffer for mammalian cells. It works with the majority of cell types and gives strong, reliable lysis. If you need to preserve native protein interactions, switch to an NP-40 or Triton X-100 based buffer instead.
RIPA Buffer Recipe (Denaturing, General Purpose)
To prepare 100 mL of RIPA buffer:
- Tris base: 790 mg (10 mM final concentration)
- NaCl: 900 mg (150 mM final)
- NP-40: 10 mL of a 10% stock (1% final)
- Sodium deoxycholate: 2.5 mL of a 10% stock (approximately 1% final, though some protocols use 0.25%)
- SDS: 0.1% final
Dissolve the Tris base in about 75 mL of distilled water first. Add the NaCl and stir until everything is dissolved. Adjust the pH to 7.4 using hydrochloric acid. Then add the NP-40 and sodium deoxycholate, stirring until the solution is clear. Finally, add SDS from a 10% stock to reach 0.1% and bring the total volume to 100 mL with distilled water.
RIPA contains three different detergents working together. The NP-40 is a mild, non-ionic detergent that disrupts cell membranes. Sodium deoxycholate is a bile salt that helps solubilize membrane-associated proteins. SDS is a strong ionic detergent that denatures proteins by breaking protein-protein interactions. Together, they lyse cells thoroughly and solubilize most protein types, but they will disrupt native protein complexes.
NP-40 Buffer Recipe (Non-Denaturing)
For experiments like co-immunoprecipitation, where you need proteins to stay folded and bound to their partners, use a gentler formulation:
- Tris-HCl, pH 7.4: 50 mM
- NaCl: 150 mM
- NP-40: 1%
NP-40 is a non-ionic detergent that compromises cell membrane integrity without denaturing proteins. It’s good for isolating cytoplasmic proteins but does not efficiently release nuclear proteins. If you need nuclear contents, you’ll need a stronger buffer or a separate nuclear extraction step. You can substitute Triton X-100 at the same concentration for a very similar result, as both are mild, non-denaturing detergents.
Bacterial Lysis Buffer
Bacterial cells have a rigid peptidoglycan wall that detergents alone can’t fully penetrate. You need lysozyme, an enzyme that degrades this wall, at a final concentration of 0.1 to 1 mg/mL. A typical bacterial lysis buffer also includes EDTA at 5 mM (to destabilize the outer membrane by chelating divalent cations) and DTT at 5 mM (to break disulfide bonds and maintain a reducing environment).
Resuspend your bacterial pellet in the buffer, incubate on ice for 20 to 30 minutes, and then either sonicate or freeze-thaw to complete lysis. After lysis, EDTA can be reduced to 2.5 mM if downstream applications are sensitive to it.
What Each Component Does
Every lysis buffer has the same core logic: break open the membrane, stabilize pH, control ionic strength, and protect your target molecules from degradation.
Tris-HCl maintains a stable pH, typically between 7.4 and 8.0. Proteins denature and enzymes behave unpredictably at extreme pH values, so this is non-negotiable. Ten to 50 mM is the standard range.
NaCl at 150 mM mimics physiological salt concentration, which prevents non-specific ionic interactions between proteins. Lower salt lets more proteins stick together nonspecifically; higher salt (up to 500 mM) can reduce aggregation and improve yield for certain applications, but may interfere with downstream enzymatic steps.
Detergents do the actual work of disrupting cell membranes. They insert into the lipid bilayer and break apart lipid-lipid, lipid-protein, and protein-protein interactions. The key distinction is strength. SDS is an anionic detergent with high affinity for proteins that denatures them quickly, making it unsuitable for sensitive protein extraction but excellent for total protein solubilization. Non-ionic detergents like Triton X-100, NP-40, and Tween 20 are milder and preserve native protein structure. Zwitterionic detergents like CHAPS carry both positive and negative charges, netting to zero, and are also mild enough for protein isolation.
Protease and Phosphatase Inhibitors
The moment you break open a cell, you release proteases that will immediately start chewing up your proteins. Adding inhibitors is essential for any protein extraction.
Commercial protease inhibitor cocktails are the most convenient option. A typical cocktail contains a broad-spectrum serine protease inhibitor, plus aprotinin, bestatin, leupeptin, pepstatin A, and a cysteine protease inhibitor, covering multiple protease classes simultaneously. One milliliter of a standard cocktail is designed to protect roughly 100 mL of lysate from 20 g of tissue.
PMSF is a commonly used serine protease inhibitor that you can add alongside a cocktail for extra coverage. One important caveat: PMSF has a half-life of only 55 minutes in aqueous solution at room temperature and pH 7.5. Add it fresh, right before use, and don’t rely on buffer you made yesterday to still have active PMSF in it.
If you’re studying phosphorylated proteins, add phosphatase inhibitors (sodium orthovanadate, sodium fluoride) as well. These prevent phosphatases from stripping off the phosphate groups you’re trying to detect.
How Much Buffer to Use
The ratio of buffer to cells matters for protein concentration. For mammalian cell pellets, a common starting point is 100 to 200 µL of lysis buffer per one million cells. Using an SDS-based buffer at 100 µL per million cells typically yields protein concentrations of 500 to 1,500 µg/mL. A modified RIPA protocol may call for 200 µL per million cells because the additional volume helps with solubilization.
If you’re working with fewer than one million cells, aim for roughly 10,000 cells per µL of lysis buffer. Too much buffer dilutes your protein below detection thresholds; too little leaves lysis incomplete and protein stuck in debris.
For tissue samples, a common ratio is 1 mL of buffer per 50 to 100 mg of tissue. Homogenize the tissue mechanically first (using a Dounce homogenizer or bead mill), then let the buffer work.
The Lysis Workflow
Start with a cold cell pellet. Wash it once in cold PBS to remove residual media and serum proteins. Add ice-cold lysis buffer with freshly added protease inhibitors. Resuspend the pellet by pipetting gently (for non-denaturing buffers) or vortexing briefly (for denaturing buffers). Incubate on ice for 15 to 30 minutes, flicking or inverting the tube every 5 minutes to keep things mixed.
After incubation, centrifuge at high speed (typically 14,000 x g for 10 to 15 minutes at 4°C) to pellet insoluble debris, membranes, and DNA. Transfer the supernatant, which is your clarified lysate, to a fresh tube. This is what you’ll quantify and use.
Storage and Stability
The base lysis buffer without protease inhibitors can be stored at 4°C for weeks to months. SDS may precipitate out of solution in the cold, so warm it briefly and re-dissolve before use if you see white flakes.
Once you’ve added protease inhibitors and lysed your cells, treat the lysate as perishable. Use prepared lysates as quickly as possible. For short-term storage (up to three months), keep them at -20°C. For anything longer, store at -80°C. Avoid repeated freeze-thaw cycles, as each one degrades protein quality. Aliquot your lysate into single-use volumes before freezing so you only thaw what you need.
Matching Buffer to Downstream Application
- Western blot: RIPA buffer works well. The denaturing conditions are compatible since you’ll be adding sample buffer and boiling anyway.
- Co-immunoprecipitation: Use NP-40 or Triton X-100 buffer. You need protein complexes to remain intact.
- Enzyme activity assays: Use the mildest possible detergent (Triton X-100 at 0.1 to 0.5%, or CHAPS) to keep enzymes functional.
- DNA extraction from bacteria: Lysozyme-based buffer with EDTA, followed by an appropriate purification method.
- Membrane protein extraction: Higher detergent concentrations or stronger detergents like SDS may be needed, but this makes native studies difficult.
When in doubt, start with the mildest buffer that gives you sufficient protein yield. You can always increase detergent concentration or switch to a harsher formulation, but you can’t un-denature a protein.