s*******e
发帖数: 23 | 1 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) | c*****t
发帖数: 1084 | 2 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 s*******e 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)
| s******y
发帖数: 28562 | 3 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：)
colocalized.
【在 s*******e 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)
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