Shreffler:Pervanadate

Overview
Pervanadate is the peroxidized form of vanadate -- an oxoanion of vanadium. It acts as an irreversible inhibitor of the family of protein tyrosine phosphatases (PTPs) with an IC50 of ~0.3 μM by oxidizing a cysteine at the enzymatic active site.

Materials

 * H2O
 * HEPES, 20 mM
 * Na3VO4
 * Catalase
 * Hydrogen Peroxide (H2O2), 30%.

Procedure

 * 1) Take one tube of 100 mM sodium orthovanadate Na3VO4 (Must be white/colorless. If it shows yellow, means VO4 forms 12-mer, cannot use.)
 * 2) Prepare 20 mM HEPES adjust pH to 7.3
 * 3) Take 30% H2O2 dilute 10 times with HEPES buffer, 5 μL 30% H2O2 + 45 μL HEPES buffer, mix gently.
 * 4) Dilute 3% H2O2 10 times further with HEPES buffer, 5 μL 3% H2O2 + 45 μL HEPES buffer, mix gently.
 * 5) Add in 10 μL 100 mM Na3VO4, 940 μL H2O. Mix by gently reversing the vial. This will make up a 1 mM pervanadate stock.
 * 6) 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.
 * 7) The pervanadate is good for 2 hours.

Discussion
discuss this protocol

Contact

 * Email Wayne Shreffler through OpenWetWare