assay buffers - (May/11/2006 )
i have a few questions
1)why is 50 mM Tris–HCl used as buffer generally..... can somebody explain to me their functions.
2)why would somebody use this "new reagent (10% ammonium molybdate, 2.5 mM copper sulfate and 100 mM ferrous sulfate in 1.25 M sulfuric acid, final)" in pyrophosphatase assay.
the reason given is Chen’s reagent gives a deep blue-colored product with pyrophosphate and therefore interferes with phosphate estimation.. which i cannot understand please enlighten me..
1) tris is a relatively benign buffer. it doesn't interfere with too many reactions (it is not perfect but i won't go into detail here). it's pK allows for its use near neutral pH (just above) where most biologicals like to hang out. and its buffering capacity at 50 mM is sufficient for most purposes.
2) chen's reagent is molybdate, sulfuric acid and ascorbic acid. it is very sensitive for inorganic phosphate but is significantly interfered with by pyrophosphate. although i am not familiar with your "new reagent" i can guess, from what you wrote, that it is either much less sensitive or insensitive to pyrophosphate and can therefore be used to determine inorganic phosphate in the presence of pyrophosphate (good if you want to monitor the breakdown of pyrophosphate).