g*********n
发帖数: 2 | 1 1. I learned the molecular concept of 'IN VIVO vs IN VITRO' from this paper,
because in my study, the in vivo means animal experiments, and i fully agree
this is the 'right'way to explore biological questions, at least in the
begining.
2. It's a tough paper for me, since I am not familiar with this field, but
good paper is always good, i did like it for the way they ask questions and
design exp. logically.
3. For the 2nd question of Assasin, it is not difficult if you can purify
seperase without | r**r
发帖数: 171 | 2 If I am wrong, correct me.
For Q5, if they use CSF extract( a kind of Xenopus extract, which should be
natually mitotic extract, once you add Calcium, a good CSF extract will start
to cycle.), they can avoid the possible artifact. The problem is in their
paper they use all human proteins(separase, securin and cohesin.) If they use
CSF extract, the proteins they can analyze for MS are Xenopus proteins. What I
don't understand is why they use human proteins.
For Q6,
Nagao K, Yanagida M.
Regulating | r**r
发帖数: 171 | 3 A good CSF extract should be arrested @ metaphase.
After the addition of calcium or fertilization which causes a transient
increase in cytoplasmic calcium conc., the CSF will be inactivated, following
by APC activation, cyclin B destruction and mitotic exit. And this process is
roughly 30 min. It is easy to collect phosphorylated separase before the
addition of calcium. To collect the unphosphorylated one, like you say, you
can throw sperm chromatin into extract and monitor the progresstion by D | r**r
发帖数: 171 | 4
CDC2/cycB or Erk2 or other kinases, but they do exclude some kinase like
polo,auroraA and CaMKII by in vitro kinase assay.
they do show some data in fig2A, they mutated the catalytic site of
separase and found that separase can not be cleaved. "As the active site lies
far from where cleavage occurs, this result implies that the cleavage of
separase is autocatalyzed" Not quite convincible.
If you are interested, you can go read "anaphase specific auto-cleavage of
separase" done by same |
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