发信人: forrestgump (sleepless in Houston), 信区: Biology
标 题: Re: cross-link
发信站: The unknown SPACE (Tue Apr 23 19:20:09 2002) WWW-POST

I think Pierce has a very comprehensive guide for protein crosslinking
reagents. did you find anything on their website? I found almost every detail
that I needed for my exp.
however, crosslinking is a tricky thing in that it varies from protein to
protein, and the condition for crosslinking matters a lot too. if you are
doing it in the presence of crude cell lysate, i doubt if there is established
protocols to follow. you probably need to try different crosslinking reagent,
different concentrations of the reagent, a time course, and various
concentration of your bait protein.
I will use the available protocol for crosslinking purified proteins in
solution by the same reagent to start with, which is probably available from
Pierce or somewhere else.
BTW, do you really need crosslinking? pull-down assays usually work quite well
without crosslinking. plus, what if the endogenous target proteins are already
saturated by endogenous bait protein?



【 在 citieeg (随波转薄好似客游) 的大作中提到: 】
: i am about to do some protein crosslink experiment.
: I will use my protein to treat the cells and then
: carry out crosslink and then to pull down the
: protein complex for further study.
:
: Since I have not done any such sort of expt b4.
: Can anybody sgiv eme some tips on this?
: the the most important is , usually, how long
: should I treat the cell, and how long i should
: apply the cross-link reagent (BS5) from pierce.
:
: I searched for protocol online, but there si not
: such protocol on it.
:
: Thanks.
:
: //bow
:

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