pyrophoshatasing cell extract for nucleotide depletion?
graumann at inky.caltech.edu
Tue Aug 6 14:09:25 EST 2002
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D.K. <dk at no.email.thankstospam.net> wrote:
>You know, only now I realized how badly I mixed it up :(
>In fact, everything I wrote in my original reply is wrong and
>the exact opposite is correct: kinases can, albeit poorly,
>thiophosphorylate, but phosphatases cannot take thiophosphate
>off. (Yes, this time I got it right for sure :-))
>So sorry! Hexokinase part is still OK, though.
This means I could (if I stick with it) pyrophosphatase as well as
hexokinase I suppose ... ;0)
>>> 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.
>IMHO, this will work only if you are very lucky...
I'm aware of this but this is a pet project, so I'm just going for it
>>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)
>No, it's not weird.
>IMO, in this case you really don't need to bother with ATP depletion.
>Even if you manage to keep all cellular ATP intact, it still will be
>diluted way below ATPgS level that you can easily provide (~ 2 mM).
I will try that.
>Just to be sure you can desalt extract over spin or gravity column -
>any commercial or home made G-25 or P-6 will do. No need to complicate
>things by introducing other stuff.
Why should the column step be necessary - someone else proposed a
depletion from nucleotides by size exclusion (see other post in thread),
but I don'tlike that idea. Looking for interactions I want to be quick
about the depletion without messing up the protein stoechiometry by
dilution even more than I do with the extraction buffer anyway ...
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