klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Fri Feb 2 12:38:50 EST 2001
seanpat at fmed2.uncu.edu.ar (Sean Patterson) wrote:
:>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
Sean, thanks for the info! Any chance you know what is temperature
and pH dependence for this reaction? 55C is not great for many proteins
(should be OK for this particular one, though). I am wondering if
30C o/n at pH > 8 will do the trick.
:What is your assay for successful palmitoylation of
This particular guy shows difference in mobility on SDS PAGE.
:Ditto that. What is your protein and why do you need to palmitoylate in vitro?
Sounds like the protein is SNAP25. It is 4X palmitated and strongly
periferal membrane bound in vivo. It is easy expressable in E.coli
in huge amounts, but without lipids it does not display all the properties
of native protein.
One thought on separation of lipidated protein from non-modified:
dialyse out octyl glucoside -> lipidated form precipitates and
since the protein is small and known to be largely unstructured
the precipitate can likely be redissolved in detergent.
Further fractionation to separate 1,2,3,4 palmitoyl might be
possible with HIC (assuming there is a sorbent that will bind in
the presence of detergent).
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