发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: MCBJC, cell division (discussion?)
发信站: The unknown SPACE (Wed Jan 22 20:34:49 2003), 站内信件

come on, show time, folks. so should we all email assassin back and corrupt
his mail box? i won't discuss all of the questions listed by assasin. actually,
to read papers on Cell is always headache, particularly manuscripts by
biochemistry/cell biology people.

: 3. In Fig. 2 A, lane 5-8, they used in vitro translated (5,6) and purified
: endogenous human SCC1 as substrates for seperase. Why the gels shows multiple
: bands besides the bands of cleaved and uncleaved hSCC1? Do you think this is a
: neat experiment? How to solve this problem?

Fig2A shows that purified separase can utilize in vitro translated(IVT) hSCC1
as well as endogenous hSCC1 as substrate in an in vitro cleavage assay. I think
the IVT sample (in RRL?) should be purified first (avoid incomplete translation),
there should be a mock w/o separase (don't like otherwise degradation though),
and include a control hSCC1 that could not be cleaved (i am not satisfied by
the Cys/Ser mutation of separase). the left side is autoradiography, sometimes
hard to avoid showing those multiple bands; the right side is western, there
are also some bands (maybe non-specific), or maybe intermediate cleavage
products, however, i do not see a clear cleavage band on the top here (compare
lane 5 and lane 7). don't know the source of the antibody.

btw, i have a question,. in Fig5A , how did they get the "separation%"?

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