I would like to test the impact of a protein on the inhibitory activity of a phosphatase. Is there a kit for assaying phosphatase activity? Or another method ?
An assay for inorganic phosphate is typical. An example is an absorbance-based assay with a molybdate reagent. Commercially-prepared reagents containing ammonium molybdate and malachite green in hydrochloric acid are available (e.g. from Biomol). The sensitivity for phosphate can be as low as the single-digit micromolar.
Hi Helene, in addition to Adam's suggestion, there are fluorescence phosphatase assay kits available from Life Technologies:
http://www.lifetechnologies.com/order/catalog/en/US/adirect/lt?cmd=catDisplayStyle&catKey=129508&filterDispName=Phosphatase%2BAssay%2BKits&_bcs_=H4sIAAAAAAAAAM1PQWrDMBB8jS41MbIFqa9u3ZQQKIXQ3oW8iQWSFaR1jPr6jhsCOYTSY2GZ0e4O%0A2plVJWTzHkM%2FGU6FqNfFnuLZGkq%2FzAfmk1CtqDeoeZ5LZw%2FEZIYxuHC0lEoTPFZTAtAIGIInUIg9%0AxXJg7%2FCLqNVSsuE40dLLRwmqZAVlU8vqAd3OXs5rj4tPRZuSzn%2B3oNpGXs%2BCjWaN7dXUxx6gexvJ%0AMF6OhdoY3wvVbT9f3yDubDo5nZ810zHEDBsY7ihDsfi7zXDQLt0NAarU%2BhLmZfzKnopuMco2jLfB%0AMDhbzv8n4Y%2FrexG%2FAY0HLdwzAgAA
(whoah...that is one long a** link!)
You could use para-nitro phenyl phosphate (Sigma) as a substrate. Carry the reaction in Tris-buffer as a function of pH (between 5.0 to 8.0) in presence of I mM Mg. The pNPP is hydrolysed to p-No2 Phenol (yellow color), the absorption is measured. The assay will identify various pH-effects and possibilities for your material. Check out the method I used for neutral K-stimulated pNPPase assay in gastric and kidney membranes.
Hi,
It is better to use various concentrations of substrate (containing p-nitrophenol phosphate). I suggest to use close range of concentrations at the beginning. Even, pay close attention to time of reaction.
- Badges
- Science topic
- Similar topics
- Cell Biology
- Methods