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Biology版 - 求助: 大家有没有遇到过这样的问题?
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相关话题的讨论汇总
话题: ip话题: protein话题: proteins话题: domain
进入Biology版参与讨论
1 (共1页)
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
3
什么意思,能说的详细点吗?
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

相关主题
为啥做overexpression却检测不到蛋白过量表达?Re: 请问用什么方法证明蛋白与蛋白的直接作用?
急问一个关于ChIP的问题蛋白哪儿去了,在线等
overexpressed的蛋白 应该transfect多久后看half life?奇怪的实验结果
进入Biology版参与讨论
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
20
去做BINDING进一步CONFIRM!
m***n
发帖数: 337
21
你是说用biocore之类的方法confirm,right?
1 (共1页)
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相关话题的讨论汇总
话题: ip话题: protein话题: proteins话题: domain