Base Ingredients:

• Tris-HCl (buffering agent prevents protein denaturation)
• NaCl (salt prevents non-specific protein aggregation) br> NP-40 (non-ionic detergent to extract proteins; 10% stock solution in H₂O)
• Na-deoxycholate (ionic detergent to extract proteins; 10% stock solution in H₂O; protect from light)

RIPA Protease Inhibitors

• Phenylmethylsulfonyl fluoride (PMSF) (200 mM stock solution in isopropanol; store at room temperature)
• EDTA (calcium chelator; 100 mM stock solution in H₂O, pH 7.4)
• Leupeptin (store frozen in aliquots, 1 mg/ml in H₂O)
• Aprotinin (store frozen in aliquots, 1 mg/ml in H₂O)
• Pepstatin (store frozen in aliquots, 1 mg/ml in methanol)

• Activated Na₃VO₄ (200 mM stock solution in H₂O; see Sodium Orthovanadate Activation Protocol)
• NaF (200 mM stock solution: store at room temperature)

Procedure

Prepare 100 ml modified RIPA buffer as follows:

1. Add 790 mg Tris base to 75 ml distilled H2O. Add 900 mg NaCl and stir the solution until all solids are dissolved. Using HCl, adjust the pH to 7.4.

2. Add 10 ml of 10% NP-40 to the solution.

3. Add 2.5 ml of 10% Na-deoxycholate and stir until solution is clear.

4. Add 1 ml of 100 mM EDTA to the solution. Adjust the volume of the solution to 100 ml using a graduated cylinder. Store RIPA buffer at 2–8°C until ready to use.

5. Ideally, the remaining protease and phosphatase inhibitors should be added to the solution on the same day you are running the assay (100 µl of aprotinin, leupeptin, and pepstatin; 500 µl of PMSF, Na₃VO₄, and NaF), but with the exception of PMSF the diluted inhibitors are stable in aqueous solution for up to 5 days. PMSF is extremely unstable in aqueous solutions, with a half-life of approximately 30 minutes, and it should be added immediately before use.

The final concentrations in the modified RIPA buffer should be:

· Tris-HCl: 50 mM, pH 7.4
· NP-40: 1%
· Na-deoxycholate: 0.25%
· NaCl: 150 mM
· EDTA: 1 mM
· PMSF: 1 mM
· Aprotinin, leupeptin, pepstatin: 1 µg/ml each
· Na₃VO₄: 1 mM
· NaF: 1 mM