发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: About protein size, any suggestion?
发信站: The unknown SPACE (Wed Jun 26 14:05:38 2002), 站内信件

is there IP assay available? i think it is necessary if you
can fractionate the protein by columns (many biochemcal stuffs
that i am not familiar, which i read from purification of
p300 transcription factor, i think people in this area knows
a lot). then do the assay to see which fraction has the
activity (is this specific reaction?) thus you know the effective
size.
is this endogenous or expressed protein? guess it is
endogenous, given the big size. try IP with the antibody and
do the assay. or IP and then fractionate it, if you have enough
material.
but this still does not solve the problem of size change.
would you run a non-reducing denaturing gel, as suggested by
burgen. unless you have an expressing clone with some tag,
you may not be able to get enough material. thinking about
SREBP, they had to purify the factor from 1000 liters of
cultured yeast 20 years ago. in this sense, you are lucky:).
【 在 aoexy (lili) 的大作中提到: 】
: Sorry I didn't say it clearly about:" why dose the protein size change?"
: Actually my protein is presumablly a protein with oxidase and ferroxidase
: activity.
: I run the protein with SDS-gradient GEL, then renaute it in glycerol. After
: that I put it into PPD(substate of oxidase). Two bands apprear on the gel,one
: of them is about 75K, the other is 50K. this protein deduced size should be
: 180k.
: Since I run SDS gel first, I think it's size won't be changed for its
: reconformation. If there is proteolysis, the total size added up should be
: around 180K.
: Any suggestion will be appreciated very much.


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