seanpat at fmed2.uncu.edu.ar
Fri Feb 2 11:47:35 EST 2001
>In article <3A768700.BE5D1F43 at slac.stanford.edu>, Mark Bowen
><mbo at slac.stanford.edu> wrote:
>:Does anyone have experience with in vitro palimoylation? I've tried the
>:reaction by adding palmitoyl CoA in DMSO to protein (25 kDa with 4 free
>:cys) in the presence of 1% octylglucoside. This doesn't seems to be
>:working based on SDS-PAGE.
Lot's of experience. I mix 3H-palm-CoA (readily soluble in water)
with my protein(s) of interest and incubate at 55C for an hour with
some detergent so there isn't a solubility problem after
palmitoylation. What is your assay for successful palmitoylation of
>Just out of curiosity - SNAP-25?
>In any case, if this reaction proceeds with any reasonable rate
>spontaneously, I am surprised, I did not know it. I was always
>under impression that you need to have some source of palmitoyl
>transferase activity. This certainly is the case for other lipid
>modifications like myristoylation and geranyl geranylation.
Yup. But with the activated donor - acyl-CoA - you can get chemical
transfer to protein thiols. This has been documented extensively in
the literature, to the point that some people are now arguing that
cellular protein palmitoylation is a non-enzymatic process.
>If you express your protein as a fusion with something, reisolation
>of it after in vitro palmitoylation in the presence of crude
>extract should be relatively straightforward thing to do.
> - Dima
Ditto that. What is your protein and why do you need to palmitoylate in vitro?
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