How To Make RIPA Lysis Buffer

About RIPA lysis buffer

RIPA, which stands for radioimmunoprecipitation assay, buffer is a commonly used lysis solution used lyse cells and tissues while preventing protein degradation. Thus, RIPA lysis buffer is used to extract proteins for their analysis, such as in Western blot or ELISA experiments.

RIPA lysis buffer works by solubilizing the cellular and nuclear membranes, via the actions of the harsh detergents sodium deoxycholate and SDS, as well as the milder NP-40. The actions of these detergents result in the breakdown of the lipid membranes, as well as protein-protein interactions, to release proteins in solution.

Don’t forget to add protease inhibitors to the RIPA lysis buffer solution to prevent any protease enzymes from prematurely digesting your proteins!

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RIPA lysis buffer recipe

The recipe below can be used to prepare a 100 mL RIPA lysis buffer solution. Scale the volumes as needed.

Reagent Volume Final concentration
Sodium chloride (5 M) 3 mL 150 mM
Tris-HCl (1 M, pH 8.0) 5 mL 50 mM
Nonidet P-40 1 mL 1 %
Sodium deoxycholate (10 %) 5 mL 0.5 %
SDS (10 %) 1 mL 0.1 %
ddH2O Up to 100 mL

How to make a RIPA lysis buffer solution

  1. Measure out 3 mL sodium chloride (5 M), 5 mL Tris-HCl (1 M, pH 8.0), 1 mL nonidet P-40, 5 mL sodium deoxycholate (10 %), 1 mL SDS (10%) and add to a 100 mL Duran bottle.
  2. Top up the Duran bottle to 100 mL with ddH2O.
  3. Mix the reagents by adding a magnetic flea into the bottle and placing on a magnetic stirrer.
  4. [Optional] Before using RIPA lysis buffer, add the desired amount of protease inhibitors (such as PMSF) to the solution.

Storage of RIPA lysis buffer

Store RIPA lysis buffer solution in the fridge (+2oC – 8oC) for relatively short periods (a few weeks).

Sometimes the detergents in the RIPA lysis buffer may re-precipitate over time. If this happens, heat the solution (37oC) and mix to dissolve the components.

For longer storage periods, aliquot and store RIPA lysis buffer in the freezer (-20oC). Thaw aliquots with gentle heat (37oC) and mix the components back into solution. Do not re-freeze once thawed.

Safety

Be careful when handling the detergents (nonidet P-40, sodium deoxycholate and SDS) in the preparation of RIPA lysis buffer, since these are hazardous. Always refer to their individual safety data sheets before using to ensure adequate protection.

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