Time to prepare
- HEPES, 20 mM
- Na3VO4 (in freezer)
- Catalase (in freezer)
- Hydrogen Peroxide, or H2O2, 30%.
Preparation of pervanadate (Huyer G et al., JBC 1997)
- Take one tube of 100mM sodium orthovanadate Na3VO4 (Must be white/colorless. If it shows yellow, means VO4 forms 12mer, cannot use.)
- Prepare 20mM HEPES adjust PH to 7.3
- Take 30% H2O2 dilute 10 times with HEPES buffer, 5 ul 30% H2O2 + 45 ul HEPES buffer, mix gently.
- Dilute 3% H2O2 10 times further with HEPES buffer, 5 ul 3% H2O2 + 45 ul HEPES buffer, mix gently.
- Add in 10ul 100mM Na3VO4, 940ul H2O. Mix by gently reversing the vial. This will make up a 1mM pervanadate stock.
- After 5 minutes, use the tip of pipet to scoop a small amount of catalase, mix in to the pervanadate stock. This will result in a burst of O2 bubbles. Open the lid of the tube to release the air pressure.
- The pervanadate is good for several hours (<24 hours).