WangLab:Pervanadate: Difference between revisions
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=== Preparation of pervanadate (Huyer G et al., JBC 1997) === | === Preparation of pervanadate (Huyer G et al., JBC 1997) === | ||
# Take one tube of 100mM sodium orthovanadate Na<sub>3</sub>VO<sub>4</sub> (Must be white/colorless. If it shows yellow, means | # Take one tube of 100mM sodium orthovanadate Na<sub>3</sub>VO<sub>4</sub> (Must be white/colorless. If it shows yellow, means VO<sub>4</sub> forms 12mer, cannot use.) | ||
# Prepare 20mM HEPES adjust PH to 7.3 | # Prepare 20mM HEPES adjust PH to 7.3 | ||
# Take 30% H<sub>2</sub>O<sub>2</sub> dilute 10 times with HEPES buffer | # Take 30% H<sub>2</sub>O<sub>2</sub> dilute 10 times with HEPES buffer |
Revision as of 11:35, 5 June 2007
Time to prepare
10 minutes
Ingredients
- H2O
- 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
- 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).