发信人: sunnyday (飞鸟), 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 01:35:59 2003) WWW-POST

呵呵，这些都不是绝对的证据。
FRET,除了你说的那个浓度的问题之外，更大的问题是做荧光标志时能否保证
不影响分子的性质和分子之间的相反作用。浓度其实反而不是特别大的问题，
只要浓度不是大得特别离谱，跟生理条件就不可能差得太远。比方说，细胞
浓度测出来是uM，只要你不超过这个量级的10倍，应该不会造成严重的后果。
至于你说的那个分子定位的问题，我觉得那就是另外一个问题了。FRET求证
的仅仅是在那个浓度下面两个分子有无interact 的potential, 至于他们当时
是否在那里，就是另外的课题了。比方说transcrpt factor 与DNA的关系。

不过没有什么东西是一定的啦。就算用了x-ray chrystagraphy证明了两个分子确实
bind在一起, 还是可以挑剔说结晶的条件跟生理条件不一定一样 ：)
要发文章时至少得有两个以上的方法吧，一般就做 IP 和2-hybrid，多数reviewer
还是会认可的：)

【 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
: : 【 在 mrp (mrp) 的大作中提到: 】
: : : 2-bybrid
:
:




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※ 修改:·sunnyday 於 Apr 13 01:35:59 修改本文·[FROM: 128.227.]
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.227.]