ATP assay - malachite green - (Feb/28/2011 )

Hi everybody!

I'm working on a ATPase protein and so i need to measure his enzymatical activity.
I red in a paper a protocol which uses the malachite green phosphate detection kit (R&D) to quantify the ATPase activity. So I command the kit and prepare similaire buffer.
But, sensibility of the kit is too high. I can't measure anything.
I hear about phosphate contamination...
Could you give me some advices...
Thank you

in any atpase assay you have to account for free phosphate from decomposing atp. running proper blanks should take care of that (we had to time our readings, including the blanks, due to the continuous decomposition of atp).

if you find the malachite green method too sensitive then you can either scale down the atpase reaction to stay within the sensitivity limits or switch to a less sensitive method.

the least sensitive may be the fiske-subbarow method.

we used micro-marsh and chen, as well as the malachite green methods but they are all within the same sensitivity range.

Thanks for your answer!
Maybe is my question a little silly but what could I do to avoid free phosphate? I'm working with gloves, new tips, new tubes, milliQ water,...
Could I sterilize everything I'm working with?
In the other hand, the methods you talk about are not use in our lab and I found nothing about it :s

no need to sterilize (don't autoclave atp). most of the free phosphate is coming from decomposition of atp.

make sure that your glassware and plasticware is not contaminated, especially with detergents (residue from "phosphate-free" detergents will saturate the assay).

the chen method: chen, ps, toribara, ty, and warner, h, anal chem, 28: 1756-8 (1956)

the marsh method: marsh, bb, biochim biophys acta, 32: 357-61 (1959)

and the fiske-subbarow method: Fiske, C. H., and Subbarow, Y., J. Biol. Chem., 66, 382 (1925).

and there is another method that i found while getting the fiske-subbarow reference: