Protein extraction & lysis
RIPA Buffer Recipe
Standard RIPA lysis buffer recipe for total protein extraction from mammalian cells. Includes components, working concentrations, protease inhibitor notes, and common mistakes.
10x RIPA stock recipe (make 10 mL, dilute 1:10 before use)
- 1 M Tris-HCl pH 8.0
- 5 mL/10 mL stockfinal 50 mM in 1x
- 5 M NaCl
- 3 mL/10 mL stockfinal 150 mM in 1x
- NP-40 (or Igepal CA-630)
- 10 mL/10 mL stockfinal 1% in 1x
- 10% sodium deoxycholate
- 5 mL/10 mL stockfinal 0.5% in 1x
- 10% SDS
- 1 mL/10 mL stockfinal 0.1% in 1x
- Nuclease-free water
- to 10 mLbring to final volume
Preparation steps
- 1Combine Tris-HCl pH 8.0, NaCl, NP-40, sodium deoxycholate, and SDS in a 15 mL tube.
- 2Bring to 10 mL final volume with nuclease-free water and mix gently — avoid foaming from the SDS.
- 3Aliquot and store at 4 °C for up to 6 months, or freeze at −20 °C for longer storage.
- 4Before use, dilute 1:10 to 1x RIPA and supplement fresh with protease inhibitor cocktail (and phosphatase inhibitors if preserving phosphorylation status).
- 5Lyse cells on ice: add 100–300 µL 1x RIPA per 10⁶ cells, incubate 30 min on ice with occasional vortexing, then centrifuge at 14 000 × g for 15 min at 4 °C.
Worked example
- To lyse a T-75 flask of adherent HEK293 cells (~5×10⁶ cells), add 500 µL of 1x RIPA supplemented with 1x protease inhibitor cocktail.
- Incubate 30 min on ice, scrape cells, transfer to a microcentrifuge tube, and centrifuge 14 000 × g, 15 min, 4 °C.
- Collect the supernatant (total cell lysate) and quantify protein by BCA or Bradford assay before loading on SDS-PAGE.
Safety and verification
- Work on ice to minimize protein degradation — always pre-chill tubes and centrifuge.
- Add protease inhibitors immediately before use; do not add to the stock.
- SDS precipitates at low temperatures; if the buffer cloudifies, warm briefly to room temperature before use.
- Sodium deoxycholate is incompatible with BCA assay at high concentrations — use a diluted sample or Bradford assay if DOC interferes.
Common mistakes
- Skipping protease inhibitors — proteases degrade your proteins within minutes of lysis.
- Lysing too many cells in too little buffer — crowded samples give poor protein solubility.
- Centrifuging at room temperature instead of 4 °C — proteolysis accelerates rapidly above ice temperature.
- Freezing the lysate with SDS and thawing slowly — SDS precipitates upon freeze-thaw; mix thoroughly after thawing.
- Using RIPA for native co-immunoprecipitation — the SDS/DOC denature protein complexes; use a milder NP-40 or CHAPS lysis buffer for co-IP.
Frequently asked questions
What is RIPA buffer used for?
RIPA buffer is used to lyse mammalian cells and solubilize total protein for western blotting, ELISA, and co-immunoprecipitation. The detergent mixture (NP-40, deoxycholate, SDS) disrupts cell and nuclear membranes efficiently.
Do I need to add protease inhibitors to RIPA buffer?
Yes — always add a protease inhibitor cocktail to 1x RIPA immediately before lysing cells. Without inhibitors, endogenous proteases degrade your target proteins within minutes.
How much RIPA buffer per cell pellet?
A general starting point is 100–300 µL of 1x RIPA per 10⁶ cells. For a T-75 flask (~5–10×10⁶ cells), use 300–500 µL. Scale by cell number, not by flask size.
Can I use RIPA buffer for co-immunoprecipitation?
RIPA contains SDS and sodium deoxycholate, which denature proteins and disrupt most protein–protein interactions. For co-IP, use a milder lysis buffer (NP-40-only or CHAPS-based) that preserves native complexes.
Is NP-40 the same as Igepal CA-630?
Yes — Igepal CA-630 is the common replacement for NP-40, which is no longer manufactured in its original form. They are chemically equivalent for most lysis applications.
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