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Na+/K+ ATP ase Assay

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#1 FlicFlak



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Posted 07 July 2009 - 06:02 AM

Hi everyone, first post!! Long time reader though.
Was just wondering if anyone has done the above assay?
The protocol im using states:
To determine the amount of phosphate produced, add 1.5 ml of a freshly prepared solution containing 9.6% (w/v) FeSO4.7H2O and 1.5% (w/v) ammonium heptamolybdate in 0.66 M H2SO4 to both standards and samples.
When i make it up in the order i get a blue/black solution which is unusuable but if i reverse it (add the molybdate then add the iron sulfate) i get a copper brown solution which i then add to my samples (Brush border membrane vesicles and Homogenate) and this detects the inorganic phosphate which give a blue colour.
My question is, why does it make a difference which substance is added first and has anyone else come accross this?

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