| Plasma Membrane ATPase Assay 【Yale University 】 | 点击： 作者：51protocol收集 来源： 时间： 2007-03-18 本站论坛
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|  | 1. Preincubate 200µl of ATPase mix at 37oC in a microfuge tube. ATPase mix made as described
below.
2. Start the reaction by adding sample. I use 50µl of a gradient fraction. Do duplicate samples so
you can average the result and do another tube that also contains 100µM vanadate. So three
samples for each fraction.
3. Incubate for 10 min at 37oC.
4. Stop reaction by adding 40µl of 50% TCA (cold) and immediately place on ice.
5. Centrifuge for 3 min in microfuge in the coldroom.
6. Remove 200µl of supernatant and place in 13 x 100mm glass tubes. The remainder can be
performed at room temp.
7. Add 1 ml of ammonium molybdate and votex tubes. Let incubate for 10 min.
8. Add 1 ml of semidine and vortex. Let incubate 20-30 min.
9. Read A710nm. Blank the spec with sample buffer alone in a reaction tube.
10. Average the two values for the sample and subtract out the value of the ( ) vanadate tube. This
gives you the vanadate-sensitive ATPase value. This is compared to the standard to get the nmoles
of phosphate released.
ATPase Mix: For 20 ml:
5mM ATP 55.12 mg
5mM MgCl2 20.4 mg
10mM PIPES 69.26 mg
5mM NaN3 200µl of a 0.5M stock
5mM phosphenolpyruvate, 20.61 mg
monopotassium salt
Adjust to pH 6.7
Can be frozen in aliquots at this point. Before using in the reaction, add 5µl per ml of Sigma
Type II pyruvate kinase to the mix.
Ammonium molybdate:
Make a 10mM stock in water and store in the dark at room temp. For 200 mls use 2.472 g of
ammonium molybdate.
Semidine:
Semidine is really 4-Aminodiphenylamine. The stock is made by mixing 200 mg into 400 ml
of 1% NaHSO3 (4g sodium bisulfate in 400 ml water). The semidine does not go into solution
well, so filter and store in the dark at room temp.
Notes:
1. For a standard curve, add TCA to the same concentration as in the samples to allow formation of
the end product. You can use a phosphate buffer to make the standard curve.
2. TCA causes slow hydrolysis of ATP which could increase the background. This is why once
the reaction is stopped the tube is immediately placed on ice.
3. The vanadate stock solution is warmed in a boiling bath a few minutes prior to use to break up
vanadate oligomers that form. The stock is stored in the dark and the solid is sodium
orthovanadate, sold by Sigma.
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