Protocol for ATPase Assay? i.e. radioactive vs non-radioactive

Kyle Legate legatek at mcmail.cis.mcmaster.ca
Sun Nov 17 11:18:45 EST 2002


On 12 Nov 2002 dustjohn at hotmail.com wrote:

>
> I am in need of a decent protocol to measure ATP hydrolysis in solution.  One way to do it is to hydrolyze radioactive ATP (cleave off the gamma-32P), use charcoal and TCA to remove protein/ATP/ADP and then scintillation count an aliquot of the supernatant.  However this method is not the greatest because there are high background levels of 32PPi in the supernatant if the ATP solution is not pure.  Does anybody have any suggestions as to what I should do?  Are there any relatively cheap non-radioactive ATPase assays?  Any ideas of a good positive control (I'm working with a Ser/Thr protein kinase so any enzyme along this line would be fantastic).
> Thanks for your help!
>
Gamma labelled ATP, TLC assay on PEI cellulose, Phosphorimager analysis.
Non radioactive assay if your protein is relatively active, do a search
for malachite green. Pi binds the dye and changes its colour properties.

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legatek at mcmaster.ca		Kyle Legate            legatek at hotmail.com

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