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Best quantitative ATPase activity assay

ATP ATPase

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#1 nimbus

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Posted 18 July 2013 - 05:06 AM

Hi everybody,

I am planning to to do experiments to measure the ATP hydrolysis activity of a protein and was wondering which way would be best. Previously, my lab used gamma-32P as well as alpha-32P labelled ATP and TLC analysis but the postdoc who started these experiments also tried the charcoal absorption assay and didn't continue it for whatever reason (she's not in the lab anymore, so I can't ask). I also found that people were using a malachite green ATPase assay which sounds very feasible and doesn't need the labelled ATP. However, I was wondering if people that have experience with ATPase activity assays could give me some feedback what worked best for them and/or what approach you would recommend.

Thanks a lot
Marco

#2 mdfenko

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Posted 18 July 2013 - 06:04 AM

i used various techniques to assay for myosin atpases.

other than a gamma-32p technique that we occasionaly used, the main difference was the method for visualizing free phosphate.

we mostly used a colorimetric method modified from chen, toribara and warner, analytical chemistry, 28, pp 1756-58 (1956). it uses ascorbic acid, sulfuric acid and molybdate to produce color that we read at 820nm.

we also used other methods (eg malachite green, marsh (similar to chen but uses citrate and more cumbersome), fiske-subbarow) but found that chen had the sensitivity and reproducibility and simplicity that we wanted.
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#3 nimbus

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Posted 19 July 2013 - 09:48 AM

Thanks a lot. Regarding the Chen protocol, after you stopped your assay do you dry your samples then in a Speedvac (or something similar) to resuspend it in H2SO4? I am asking because all the protocols I found and that adapted this method mentioned to remove all solvent before adding the H2SO4?

#4 mdfenko

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Posted 19 July 2013 - 10:55 AM

we did it a different way. we didn't dry our samples.

we ran the assay in a reaction volume of 700ul (these volumes can be scaled down if necessary). kill the reaction with 200ul 10% tca. sit on ice for a few minutes then spin in an eppendorf centrifuge for 2 minutes ( this was a long time ago, the eppendorf only went one speed then).

then we would take 750ul (out of 900, remember this for the calculation, multiply by 1.2) and add 500ul of the chen reagent (mix equal volumes of 10% ascorbic acid (made fresh), 6N sulfuric acid (18ml concentrated to 108ml with water) and 2.5% ammonium molybdate tetrahydrate). mix and incubate at 25C for 1 hour then read at 820nm. compare to a standard curve of 0-80 nmoles inorganic phosphate prepared to 750ul and treated like the samples.

make sure you include proper blanks (atpase reaction without enzyme, reaction mix without atp but with enzyme) for subtraction.

a couple of important notes:

we found that when we ran a lot of assays that we had to time the addition of chen reagent and when we read the sample (the unreacted atp will continue to decompose) we used a stopwatch to maintain the interval.

all glassware (or plasticware) used must be very clean and, if not disposable (like graduated cylinders, etc), must have been cleaned with phosphate-free detergent and thoroughly rinsed with di water. the reagent is very sensitive to phosphate and will turn if there is even a trace of phosphate present (we prepared the reagent and let it sit for a few minutes to ensure that it wasn't contaminated).

Edited by mdfenko, 19 July 2013 - 10:55 AM.

talent does what it can
genius does what it must
i do what i get paid to do





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