m***n
发帖数: 337 | 1 我在mapping一个protein (A)和另外一个protein (B)相互作用的区域。首先wt A 和B
是有相互
作用的。 我发A的predict的6个domain分别表达，然后和B co－transfection， IP显
示单独的
6个domain每个都可以和B相互作用。 我的negative和positive IP control都没问题。
结果让
我很难相信，我也不知道是什么原因。大家有没有遇到类似的问题，any suggestions
and
explanations would be appreciated. thank you so much |
b******n
发帖数: 4225 | 2 B被A包住了吧
B
suggestions
【在 m***n 的大作中提到】
: 我在mapping一个protein (A)和另外一个protein (B)相互作用的区域。首先wt A 和B
: 是有相互
: 作用的。 我发A的predict的6个domain分别表达，然后和B co－transfection， IP显
: 示单独的
: 6个domain每个都可以和B相互作用。 我的negative和positive IP control都没问题。
: 结果让
: 我很难相信，我也不知道是什么原因。大家有没有遇到类似的问题，any suggestions
: and
: explanations would be appreciated. thank you so much
|
m***n
发帖数: 337 | |
a***e
发帖数: 1010 | 4 Since you are doing co-transfection to overexpress two proteins
simultaneously, it is quite possible to bring in such false positive outcome. |
T**********t
发帖数: 1604 | 5 结果为什么很难相信？
既然你的positive和negative control都没问题，如果这些数据又能够被重复的话，你
就应该相信你的实验数据啊。你有什么根据认为A和B的interaction只发生在某几个
domain上么？ |
m***n
发帖数: 337 | 6 我没有根据A和B的interaction只发生在某几个domain。就是从来没有遇到这种情况，
老板不相信，
说实话自己也不是很sure。尽管result很clean，而且重复性很好。
【在 T**********t 的大作中提到】
: 结果为什么很难相信？
: 既然你的positive和negative control都没问题，如果这些数据又能够被重复的话，你
: 就应该相信你的实验数据啊。你有什么根据认为A和B的interaction只发生在某几个
: domain上么？
|
m***n
发帖数: 337 | 7 could you please explain a little bit on why overexpressing two proteins
simutaneously will bring in false positive? thank you so much and wait on
line.
outcome.
【在 a***e 的大作中提到】
: Since you are doing co-transfection to overexpress two proteins
: simultaneously, it is quite possible to bring in such false positive outcome.
|
a***e
发帖数: 1010 | 8 Overexpresson can increase the concentration of a protein to a degree much
higher than physiological level. Under this situation, the protein may not
be correctly processed, degraded, modified, or sorted to its destination.
And during the co-IP process, many proteins may have a chance to interact
with others if the concentration is too high.
Generally speaking, people don't favor co-overexpress two protein together
to do co-IP.
on
【在 m***n 的大作中提到】
: could you please explain a little bit on why overexpressing two proteins
: simutaneously will bring in false positive? thank you so much and wait on
: line.
:
: outcome.
|
s******y
发帖数: 28562 | 9 Sometimes the IP can be artifact of over-expression, if not, then it is
possible that your protein A has many domains that can all bind to B. This
is possible. For example, talin has more than 11 vinculin binding domains.
B
suggestions
【在 m***n 的大作中提到】
: 我在mapping一个protein (A)和另外一个protein (B)相互作用的区域。首先wt A 和B
: 是有相互
: 作用的。 我发A的predict的6个domain分别表达，然后和B co－transfection， IP显
: 示单独的
: 6个domain每个都可以和B相互作用。 我的negative和positive IP control都没问题。
: 结果让
: 我很难相信，我也不知道是什么原因。大家有没有遇到类似的问题，any suggestions
: and
: explanations would be appreciated. thank you so much
|
m***n
发帖数: 337 | 10 if this is the case, except for overexpression, is there any other good
ways I could use to do the mapping? Any input would be appreciated.
much
not
destination.
interact
together
【在 a***e 的大作中提到】
: Overexpresson can increase the concentration of a protein to a degree much
: higher than physiological level. Under this situation, the protein may not
: be correctly processed, degraded, modified, or sorted to its destination.
: And during the co-IP process, many proteins may have a chance to interact
: with others if the concentration is too high.
: Generally speaking, people don't favor co-overexpress two protein together
: to do co-IP.
:
: on
|
|
|
m***n
发帖数: 337 | 11 this is amazing. I am thinking if there is any other good methods beyond
the IP to check the protein-protein interaction, which could avoid the
overexpression problem?
This
domains.
【在 s******y 的大作中提到】
: Sometimes the IP can be artifact of over-expression, if not, then it is
: possible that your protein A has many domains that can all bind to B. This
: is possible. For example, talin has more than 11 vinculin binding domains.
:
: B
: suggestions
|
s******y
发帖数: 28562 | 12 Yesh, for example you can use IP on endogenouse proteins
For the protein domain things, you can use the recombinant domain of A to
pulldown the endogenous proteins B
【在 m***n 的大作中提到】
: this is amazing. I am thinking if there is any other good methods beyond
: the IP to check the protein-protein interaction, which could avoid the
: overexpression problem?
:
: This
: domains.
|
m***n
发帖数: 337 | 13 but in this way, I still can't avoid the overexpression of A, which is
stated by ahche" overexpression of A brings the false positive in".
to
【在 s******y 的大作中提到】
: Yesh, for example you can use IP on endogenouse proteins
: For the protein domain things, you can use the recombinant domain of A to
: pulldown the endogenous proteins B
|
s******y
发帖数: 28562 | 14 well, in this case, you can check with the other endogenous protein which is
not supposed to have interaction with A, and see if they also get pulldown.
【在 m***n 的大作中提到】
: but in this way, I still can't avoid the overexpression of A, which is
: stated by ahche" overexpression of A brings the false positive in".
:
: to
|
m***n
发帖数: 337 | 15 Could you give me some reference on the talin-vinculin interaction? thank
you very much
This
domains.
【在 s******y 的大作中提到】
: Sometimes the IP can be artifact of over-expression, if not, then it is
: possible that your protein A has many domains that can all bind to B. This
: is possible. For example, talin has more than 11 vinculin binding domains.
:
: B
: suggestions
|
m***n
发帖数: 337 | 16 yes, I will do that.
your point is: if I only overexpress one protein, it would be better than
expressing both, right?
which is
pulldown.
【在 s******y 的大作中提到】
: well, in this case, you can check with the other endogenous protein which is
: not supposed to have interaction with A, and see if they also get pulldown.
|
s******y
发帖数: 28562 | 17 Gingras AR, Ziegler WH, Frank R, Barsukov IL, Roberts GC, et al. (2005) Mapping and consensus sequence identification for multiple vinculin binding sites within the talin rod. J Biol Chem 280: 37217–37224.
Also:
http://www.sciencemag.org/cgi/content/abstract/323/5914/638
http://www.ploscompbiol.org/article/info:doi%2F10.1371%2Fjournal.pcbi.0040024
【在 m***n 的大作中提到】
: Could you give me some reference on the talin-vinculin interaction? thank
: you very much
:
: This
: domains.
|
m***n
发帖数: 337 | 18 Thank you so much, Sunnyday.
Mapping and consensus sequence identification for multiple vinculin
binding sites within the talin rod. J Biol Chem 280: 37217–37224.
http://www.ploscompbiol.org/article/info:doi%2F10.1371%2Fjournal.pcbi.0040
024
【在 s******y 的大作中提到】
: Gingras AR, Ziegler WH, Frank R, Barsukov IL, Roberts GC, et al. (2005) Mapping and consensus sequence identification for multiple vinculin binding sites within the talin rod. J Biol Chem 280: 37217–37224.
: Also:
: http://www.sciencemag.org/cgi/content/abstract/323/5914/638
: http://www.ploscompbiol.org/article/info:doi%2F10.1371%2Fjournal.pcbi.0040024
|
O******e
发帖数: 4845 | 19 People usually try overexpression co-IP to get some confidence before
playing
with endogenous proteins.
【在 a***e 的大作中提到】
: Overexpresson can increase the concentration of a protein to a degree much
: higher than physiological level. Under this situation, the protein may not
: be correctly processed, degraded, modified, or sorted to its destination.
: And during the co-IP process, many proteins may have a chance to interact
: with others if the concentration is too high.
: Generally speaking, people don't favor co-overexpress two protein together
: to do co-IP.
:
: on
|
n****n
发帖数: 165 | |
m***n
发帖数: 337 | 21 你是说用biocore之类的方法confirm，right？ |
