Ok, first off HPLC is definitely not the method of choice, because HPLC works under conditions (i.e. acetonitrile, DCM or methanol with 0.1 % TFA)
where proteins unfold, meaning that activity is lost due to loss of the native fold. Essentially you end up with a polypeptide of random
structure rather than the defined original structure of the native folded enzyme.
So you have to use buffers - usually isotonic of some kind (50 mM K phosphate, 100 mM NaCl pH 7.5 springs to mind).
Most proteins are happy under these conditions.
Maya, the procedure using ammonium sulfate precipitation, is this out of a text book referring to ADH or PDC specifically or one of many purification
protocols used for protein purifications in general? With ammonium sulfate, the target precipitates only at a certain AS concentration range, which is
the basis for purification! So usually steps of 0-0.5, 0.5-1.0, 1.0-1.5 etc M (NH4)2SO4 (or finer, upon optimisation) are taken and the fraction that
contains the protein (assayed by mass, activity, whatever) is then further purified....but this is old school biochem.
Whole cell vs purified - naturally the whole cell extract is better because it contains cofactors and other proteins that you may not have present in
your purified extract.
Despite what biologists claim about knowing about a particular subject/reaction, much more is UNknown. A fact I found out the hard way. And there are
many crap biologists/biochemists, and I freely admit they wouldn't survive in other sciences.
Anyway, I can check up ADH (alcohol dehydrogenase) and PDC (pyruvate decarboxylase) in i.e. Methods in enzymology, but in the meantime a patent
search might useful.
If you give me the biochemical properties of ADH/PDC (MW, pI, solubility, etc), I can work out a purification scheme for you...
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