发信人: Marble (小石头哥哥), 信区: Biology
标 题: MCBJC, cell division, discussion
发信站: The unknown SPACE (Sun Jan 26 01:07:48 2003) WWW-POST

【 在 assasin (冷血---何时能被加热？) 的大作中提到: 】
: Hi, folks,
:
: please read and discuss. and make this first MCBJC work.

i do not think that everybody has read this paper. we need more enthuisasm and
effort to keep active discussion and make this MCBJC run. meanwhile, think
about the paper that you will pick up later.

: : 4. In Fig. 3 B & C, they tried to purify endogenous human securin/seperase
: : complex. However, silver staining (Fig. 3 C) showed that there were other
: : proteins in fraction 5 & 6. Do you think these unknown proteins would
cause
: : problems to the in vitro cohesin cleavage assay? How to make more pure
: : securin/seperase preparatons?

these unknown proteins could interact with separase/securin and thus make the
result hard to interpret. an affinity purification might help, however, it
still
could not exclude contamination of tightly associated proteins. yet the major
goal for this part of work is to identify "inhibitory factors", particularly
phosphorylation of separase per se. i am glad that they indeed found the
inhibitory phosphorylation site by mass spec. and then later they showed that
expressed phosphomutants support their hypothesis that phosphorylation can
inhibit separase. i am wondering whether they use bacteria or mammalian
expression system. for mammalian system, my concern is that purified protein
might also associate with unknown polypeptides.



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