发信人: clonist (dream on), 信区: Biology
标 题: Re: 请问用什么方法证明蛋白与蛋白的直接作用？
发信站: The unknown SPACE (Sun Apr 13 01:24:59 2003), 站内信件

good comment. your standard is very high. but usually you can
do couple of assays and hope all give you the same positive
results. then you'll feel more comfortable to conclude that
you find the interaction you're looking for. for endogenous
staining now people are looking for 3-D re-construction to
verify true co-localization.

【 在 smileface (笑脸) 的大作中提到: 】
: yeast two-hybrid can't be used to definitely say protein A and protein B
: are directly interacted.
: To say two proteins are directly interacted, there are a lot of things
: you should take care.
: (1) yeast 2-hybrid was used to screen candit protein that may interact
: with yoru protein. A of of artificial positive exist.
: because : both in yeast 2-hybrid and mammalian 2-hybrid, you have
: overexpressed something to such an abundant extent.
: some RNA binding proteins bind bnd RNAs as a bridge.
: (2)so, you need GST-pull down, Co-IP to further test the binding,
: However, during GST-pull donw, co-IP, if you are usinge lysate from cells,
: you may have created a condition to bring together two proteins that
: locates in different places in cells, such as one in Nuc, the other in Cyt.
: (3)so, you need staining of endogenous protiens to show they are colocalized.
: (4) If the FRET case, you also meet a problem that these two proteins
: may locate in different places in cells. And may have different
: concentrations as u have used in FRET.
: So, it is not that simple to test whether two proteins can interact
: directly or not
: however, one thing should alwasy keep in mind. You have test them
: in different assays, you have right controls. and most important
: this interaction is important for their physiological funtions.
: 【 在 sunnyday (飞鸟) 的大作中提到: 】
: : hehe, typo. Yeast 2-hybrid
: : Or FRET


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