发信人: aidyzeng (xx), 信区: Biology
标 题: Re: 问一个co-IP buffer的问题
发信站: BBS 未名空间站 (Thu Apr 3 00:05:57 2008)

DTT is not included in Xenopus egg extract, as far as I'm aware of. This is
because the egg contains a lot of reducing reagent already, say, GSH, which
is maintaining a reducing intracellular environment. Normally you shouldn't
see disulfide bonds with intracellular proteins.

High concentration of K+ will precipitate with the dodecyl sulfate anion for
sure. However, if you limit [K+] below 100mM, which is a reasonable mimic
of intracellular environment, it is usually fine.

Regarding if divalent ions are required for your co-IP, if you cannot make
any prediction based on available information, the best way to go might be
trying +/- Mg2+ and Ca2+ conditions. Intracellular Mg2+ is 0.5-1mM and Ca2+
is extremely low, unless under special situations. My feeling is the chance
that you will see a difference is very small.


【 在 juicemaker (Almost there) 的大作中提到: 】
: Thanks a lot!
: What confuses me is that whether I need to add small amount of divalent
ions
: for co-IP to simulate intracellular environment.
: And as to DTT, will it introduce some artifacts by destroying the existing
: disulfide bonds or changing the redox status? I just realized that DTT is
: normally used for preparation of xenopus extracts, but have no idea if it
is
: definitely required.



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