ATP affinity chromatography
Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Sat Jun 24 10:41:37 EST 2006
Ruben Martinez Buey wrote:
> Dear all,
> Has anybody experience with ATP-sepharose /ATP-agarose chromatography?
> Is there any recommended protocol? How many times can I use the column?
> Is the ATP in the column going to be hydrolized?
I have done it with the molecular chaperone Hsc70, and in this case
hydrolysis to ADP doesn't matter, as the protein has high affinity for
that as well.
It is worthwhile to remove nucleotides from the sample, for example by
ammonium sulphate precipitation of the protein. In my case that also
removed turbidity from the sample (much easier than spinning at 100,000
g if you need to process several l of cytosol) and an apyrase activity
that would otherwise destroy the column. Then just pass the sample
slowly over the column, wash with buffer until the A280 is back to
blank, followed by a wash with as high a KCl concentration as you can
without destroying the ligand-protein association. Go back to salt free
buffer and elute with ATP-solution (1 mM). Of course Hsc70 forms a very
tight (low k_off) bond with ATP, so extensive washing is possible. Hence
it was useful to incubate the column for 10-15 min with the ATP solution
before continuing the elution. Thus one can reduce the required volume
of ATP-buffer, which is expensive.
In my hands the column could be re-used a couple of time if stored with
20% methanol in the cold, but it will eventually loose capacity. That's
a big shame, the stuff is bloody expensive.
Many ATP-binding enzymes, including Hsc70, also bind fluorescein
derivatives. It would be ideal if those could be used for
chromatography, but coupling of activated fluorescein to Lys-agarose as
spacer did not result in gels with affinity to Hsc70, regardless of
whether the link was with C-6 of the benzene ring or to C-4´ in the
tripple ring. If anybody has an idea I would be eager to learn about it.
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