发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: help! 作业问题求救
发信站: The unknown SPACE (Fri Apr 18 14:55:34 2003), 站内信件

your question does not provide enough information, at least
you should suggest its possible function, for example,
topology, intracellular domain, etc. this ligand could be
chemical compound, peptide hormone, or protein-protein
interaction activation, etc.
a couple of suggestions if it is an unknown gene:
search for homologous gene, function in model systems from
C elgans, fruit fly to mouse? what is already known?
protein motif analysis (post-translational modification?
proteolysis sites? possible interaction with other proteins?)
topology analysis (computational and experimental, type I
or type II transmembrane?)
gene expression profile (which tissue? what type of cell?)
subcellular localization (transmembrane - plasma/nuclear/
organelle?)
Y2H with extracellular domain as bait to look for protein
interactions, yet it will not solve the ligand question
(btw, Y2H is really risky, you may find too many false
positive targets). if a potential target found, search for
its involvement in other signalling pathway. siRNA
transfection assay and address the downstream signalling?
computational analysis of the extracellular domain and search
for known motif that is involved in binding to small ligand
(not reliable analysis)
set up a functional assay in which you can screen a large
chemical compound library to find potenital chemical ligand
mutagenesis of extracellular domain
if genetic screen is available for this gene in C elegans,
that is my favorite approach then.
is this a homework? tell you tutor that it is not a good
question without enough information.

【 在 wsdoll (doll) 的大作中提到: 】
: 我有一个transmembrane protein.想用yeast two hybrid 找它的extracellular domain
: 的
: 配体。
: 可是想法
: 却被老师否决
: 了。 认为这个实验可能什么都不能得到。 为什么不可行？ 我听说有一个什么expression cloning methond
: 常用来找ligand。的 可是我看不出这两种方法的区别。
: 还有
: 什么好方法找配体吗？
: 如果配体不是蛋白， 比如说， 气体， 怎么找？
: 感激不尽！


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