Common Lysis Buffers
Lysis buffers are specialized solutions used to break open (lyse) cell membranes and release intracellular contents, such as DNA, RNA, proteins, lipids, or organelles, for downstream experiments including:
- Purification
- Enzymatic assays
- In-situ hybridization (ISH)
- Western blotting
- Immunoprecipitation (IP)
- Immunohistochemistry (IHC)
- ELISA
Choosing the right buffer is crucial: it can affect yield, integrity, specificity, and the success of downstream analyses.
What is a Lysis Buffer?
A lysis buffer is a chemical solution that disrupts cell membranes (or walls) to liberate internal components. These reagents contain different chemicals specialized for degrading certain membranes. It typically includes a buffer (to stabilize pH), salts, detergents, inhibitors, and chelators.
Image 1: The mechanism of cellular lysis. Using a detergent within a lysis buffer solution, lysates may be extracted from specific cellular compartments for later analysis.
How to Choose a Lysis Buffer: Step-by-Step Guide
- Define the analyte(s) of interest
- Are you after soluble cytoplasmic proteins, nuclear proteins, membrane proteins, mitochondrial proteins, or RNA/DNA?
- Tough membranes (plant, yeast, bacteria) may need more aggressive detergents or physical lysis.
- Understand the downstream applications
- For enzymatic assays, you need buffers that preserve activity (i.e. mild, non-denaturing).
- In Western blot, immunoprecipitation, or ELISA, protein integrity and solubility matter.
- In mass spectrometry, low detergent concentration or detergent-free may be preferred.
- Balance strength vs. specificity
- Strong buffers (e.g. RIPA) extract more, but risk extracting unwanted proteins or destroying protein complexes.
- Mild buffers preserve complexes but may give lower yield or miss membrane/nuclear proteins.
- Consider additives
- Protease inhibitors (to prevent protein degradation)
- Phosphatase inhibitors (if phosphorylation status is of interest)
- Chelators such as EDTA (but note: may interfere with metal-dependent enzymes)
- Reducing agents if needed
- Check compatibility
- Detergents can interfere with downstream assays (e.g., detergents may hamper mass spec, enzyme kinetics, or membrane protein reconstitution).
- Buffer pH and salt concentrations should be compatible with downstream enzymatic reactions or antibody binding.
- Pilot test / optimization
-
- Test 2-3 buffer types in small scale to compare yield, specificity, and downstream performance.
- Adjust detergent concentration, incubation time, temperature as needed.
Common Lysis Buffers & Applications
Practical Tips & Best Practices
- Use inhibitors: Add protease and phosphatase inhibitors stock should be added just before lysis. Some degrade over time.
- Keep everything cold (on ice) during lysis and centrifugation to reduce protease activity.
- Work fast: Minimize the time between cell harvest and protein/RNA stabilization.
- Clarify lysates by centrifugation to remove debris; this reduces background in downstream analysis.
- Consider detergent removal when needed: Some assays require detergent-free samples; use dialysis, spin filters, or detergent-removing resins.
- Validate lysis efficiency: Use controls like Western blots for cytosolic vs nuclear markers or microscopy to confirm lysis.
Troubleshooting common issues:
Problem |
Possible Cause |
Suggested Fixes |
|---|---|---|
| Low Yield | Weak buffer / low detergent / insufficient incubation time | Use stronger buffer (e.g. RIPA), increase incubation, increase detergent concentration |
| Loss of protein activity | Harsh detergent / proteases / inappropriate pH | Use mild buffer, include inhibitors, optimize pH, use reducing agents only if needed and if disulfide protein linkage is not critical. |
| High background in assays | Debris not cleared / non-specific proteins pulled down | Centrifuge well; preclear; use more specific buffer; adjust detergent types |
| Membrane proteins insoluble | Wrong type of detergent; too mild | Use detergents better at solubilizing membranes (ionic or stronger non-ionic or zwitterionic) |
Boston BioProducts’ Lysis Buffer Options & Custom Formulation
- Browse our catalog of pre-formulated lysis buffers (NP-40, RIPA, CHAPS, Triton X-100, etc.), each optimized for different cell types and analyte locations.
- Use our Custom Manufacturing / Reagent Builder to design a buffer tailored to your specific needs (detergent choice, inhibitor mix, buffer strength).