pyrophoshatasing cell extract for nucleotide depletion?
graumann at sue.caltech.edu
Mon Aug 5 23:32:32 EST 2002
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In article <ain9a2$2eq$1 at news.doit.wisc.edu> you wrote:
> The best I can come up with is to use hexokinase with glucose.
> It depletes ATP levels incredibly fast, given a ~ mM glucose.
> Following this an excess of ATPgS should do the job I assume you
> need. Any residual ATP synthesized from other NTPs and/or ADP
> should not be a problem as hexokinase will remain active.
> (Doing all the right controls still applies). Hexokinase is dirt cheap
> from Sigma, BTW.
Thanks for the hint.
> If you can provide a more detailed description of the experiment
> you are trying to do, odds are good there is a better solution.
Ok, here comes the embarrasing part (I have no idea whether this is a
reasonable approach) ...
I'm setting out to look by pulldown/masspec for the substrate of a
kinase. I will try pulldown straight from extract and pulldown from
extract of cells that have been formaldehyde crosslinked. I found some
papers revferring to the stabilization of kinase/substrate complexes in
the presence of g-S-ATP (albeit only for peptides in vitro) and hence
the question above. I would make an extract, deplete it for ATP while
providing g-S-ATP and pull down my kinase, hoping that g-S-ATP has
replaced ATP and might stabilize the kinase/substrate complex. Sounds
weired? - may well be ;0)
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