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ATPase assay


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#1 swann

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Posted 06 December 2010 - 03:46 AM

Hi everybody,
I am planning to perform an ATPase assay on an IP without using any kit. I have actually found a protocol on a paper, which says to resuspend the beads from the IP in a Tris buffer containing ATP and let the reaction to proceed for 15' at 30C "prior to quantification of released phosphate". Now, how am I supposed to quantify the released phosphate? Does anybody have an idea of which kind of reaction or detection method should I use?
Thank you in advance for any suggestion

#2 mdfenko

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Posted 06 December 2010 - 07:26 AM

there are a number of colorimetric methods to determine phosphate release. we used a modification of the chen phosphate determination (p.s. chen jr., et al, analytical chemistry, 28: 1756-1758 (1956)). there's also the marsh method (b.b. marsh, biochimica biophysica acta, 32: 357-361 (1959)) and the fiske-subbarow method and one using malachite green (sorry, i don't have either reference handy).

or you can use 32p labeled atp and determine the release of radioactivity as inorganic phosphate (we did it on tlc).

keep in mind that atp is unstable under the conditions that you use to measure phosphate so the readings will continue to increase as you read them.
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