Shreffler:Pervanadate

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Contents

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

References

  1. Huyer G, Liu S, Kelly J, Moffat J, Payette P, Kennedy B, Tsaprailis G, Gresser MJ, and Ramachandran C. . pmid:8995372. PubMed HubMed [Huyer]

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