b****u 发帖数: 903 | 1 在revision,review建议endogenous的coip检验是否binding,表明真实的相互作用状态,我们文章里面,overexpression可以检测到binding,但内源的试了几次,摸索了不同buffer条件没有成功,以前就看相关文献,一篇cell里面可以内源检测到该蛋白和一个底物binding,但我们没有重复出来,就连overexpression这2个蛋白,我们也没检测到binding,哎,十分frustrated啊,coip就是很tricky。 |
A******y 发帖数: 2041 | 2 Now you know how many over-expression data are probably wrong. Do you know
the binding site? How about introduces mutation at the binding interface
and see if you still can ip it down with over-experssion. |
w***a 发帖数: 1053 | |
s******s 发帖数: 13035 | 4 //nod, 检测有没有相互作用应该用这个呀,怎么能用co-ip
再说,就算有Interaction,co-ip也不一定work
【在 w***a 的大作中提到】![](/moin_static193/solenoid/img/up.png) : GST-pull down
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a***e 发帖数: 1010 | 5 GST pull down is a more artificial assay, while co-IP more represents the
endogenous case.
【在 s******s 的大作中提到】![](/moin_static193/solenoid/img/up.png) : //nod, 检测有没有相互作用应该用这个呀,怎么能用co-ip : 再说,就算有Interaction,co-ip也不一定work
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O******e 发帖数: 4845 | 6 GST PD: direct interaction;
Co-IP: interaction, maybe indirect.
【在 a***e 的大作中提到】![](/moin_static193/solenoid/img/up.png) : GST pull down is a more artificial assay, while co-IP more represents the : endogenous case.
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C***Y 发帖数: 61 | 7
状态,我们文章里面,
overexpression可以检测到binding,但内源的试了几次,摸索了不同buffer条件没有
成功,以前就看相关文献,一篇cell
里面可以内源检测到该蛋白和一个底物binding,但我们没有重复出来,就连
overexpression这2个蛋白,我们也没检测到
binding,哎,十分frustrated啊,coip就是很tricky。
Hopefully these tips will be helpful.
Make sue you have a very good 1st antibody. A strong antibody is capable of
pulling down weak interaction
partners.
Crosslink 1st antibody to beads and elute co-purified protein complex from beads
after washing (instead of boiling
beads) to reduce background.
Use ECL plu
【在 b****u 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 在revision,review建议endogenous的coip检验是否binding,表明真实的相互作用状态,我们文章里面,overexpression可以检测到binding,但内源的试了几次,摸索了不同buffer条件没有成功,以前就看相关文献,一篇cell里面可以内源检测到该蛋白和一个底物binding,但我们没有重复出来,就连overexpression这2个蛋白,我们也没检测到binding,哎,十分frustrated啊,coip就是很tricky。
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v***a 发帖数: 1242 | 8 我也有类似的co-ip不work的困扰。
请问你说的:
“Crosslink 1st antibody to beads and elute co-purified protein complex from
beads after washing (instead of boiling beads) to reduce background”
怎么个crosslink法?我是加了1st Ab overnight再加beads的。
还有elute我是用的SDS sample buffer,然后就boil,100 C,10min。boiling beads
和elute是两个独立的过程呀,为啥elute可以取代boiling?是我理解错你的意思了吗?
关于ECL plus,我觉得用ECL曝光久一点,貌似也跟plus一样的效果,而且plus还会令
blot颜色深浅不均一,不知道你会有这个问题吗?如果有,怎么解决比较好?
谢谢回答。
of
【在 C***Y 的大作中提到】![](/moin_static193/solenoid/img/up.png) : : 状态,我们文章里面, : overexpression可以检测到binding,但内源的试了几次,摸索了不同buffer条件没有 : 成功,以前就看相关文献,一篇cell : 里面可以内源检测到该蛋白和一个底物binding,但我们没有重复出来,就连 : overexpression这2个蛋白,我们也没检测到 : binding,哎,十分frustrated啊,coip就是很tricky。 : Hopefully these tips will be helpful. : Make sue you have a very good 1st antibody. A strong antibody is capable of : pulling down weak interaction
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C***Y 发帖数: 61 | 9
from
beads
吗?
Here are the details:
Incubate 1at Ab with beads for 1 hr at RT. Wash away the unbound Ab.
Crosslink Ab to beads with Imidoester
Crosslinkers (Pierce). Incubate Nuclear extract (or whole cell lysate) with
beads-Ab.
After washes, elute the co-purified protein complex with low PH glycine
solution instead of boiling beads.
【在 v***a 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 我也有类似的co-ip不work的困扰。 : 请问你说的: : “Crosslink 1st antibody to beads and elute co-purified protein complex from : beads after washing (instead of boiling beads) to reduce background” : 怎么个crosslink法?我是加了1st Ab overnight再加beads的。 : 还有elute我是用的SDS sample buffer,然后就boil,100 C,10min。boiling beads : 和elute是两个独立的过程呀,为啥elute可以取代boiling?是我理解错你的意思了吗? : 关于ECL plus,我觉得用ECL曝光久一点,貌似也跟plus一样的效果,而且plus还会令 : blot颜色深浅不均一,不知道你会有这个问题吗?如果有,怎么解决比较好? : 谢谢回答。
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v***a 发帖数: 1242 | 10 非常感谢
还有2个问题:
1)你是用什么buffer来incubate 1st Ab 和 beads的?跟wash一样的buffer吗?我
wash是用IP lysis buffer。你会preclean nuclear extract/cell lysate吗?
2)low pH glycine solution具体是多low?6.8?这个是可以直接elute然后
centrifuge了就可以上样跑胶?
with
【在 C***Y 的大作中提到】![](/moin_static193/solenoid/img/up.png) : : from : beads : 吗? : Here are the details: : Incubate 1at Ab with beads for 1 hr at RT. Wash away the unbound Ab. : Crosslink Ab to beads with Imidoester : Crosslinkers (Pierce). Incubate Nuclear extract (or whole cell lysate) with : beads-Ab. : After washes, elute the co-purified protein complex with low PH glycine
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C***Y 发帖数: 61 | 11
PBS or washing buffer can be used for incubating Ab with beads;
I have never done preclear;
Glycine solution (pH~2.8) can be used for elution. After elution, add equal
volume of 1 M Tris, PH7.5 (for
neutralization) and equal volume of 3 X SDS loading buffer.
【在 v***a 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 非常感谢 : 还有2个问题: : 1)你是用什么buffer来incubate 1st Ab 和 beads的?跟wash一样的buffer吗?我 : wash是用IP lysis buffer。你会preclean nuclear extract/cell lysate吗? : 2)low pH glycine solution具体是多low?6.8?这个是可以直接elute然后 : centrifuge了就可以上样跑胶? : : with
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b****u 发帖数: 903 | 12 多谢各位的建议,crosslink我也曾经try过,就是重复不了别人的实验,人家内源也可
以pulldown,咱们overexpressing都不行,差距那叫一个大啊。
BTW,可能不同lab做COIP的时候的buffer条件不一样吧。 |
v***a 发帖数: 1242 | 13 多谢多谢!
equal
【在 C***Y 的大作中提到】![](/moin_static193/solenoid/img/up.png) : : PBS or washing buffer can be used for incubating Ab with beads; : I have never done preclear; : Glycine solution (pH~2.8) can be used for elution. After elution, add equal : volume of 1 M Tris, PH7.5 (for : neutralization) and equal volume of 3 X SDS loading buffer.
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b*******H 发帖数: 830 | 14 pH2.8 PLUS 1/20 V of pH9.4, or pH3.5 elution plus 1/10 V of pH8.5 could be
better. otherwise, sample going to be diluted too much.
remember doing it at RT, and no more than 15min.
equal
【在 C***Y 的大作中提到】![](/moin_static193/solenoid/img/up.png) : : PBS or washing buffer can be used for incubating Ab with beads; : I have never done preclear; : Glycine solution (pH~2.8) can be used for elution. After elution, add equal : volume of 1 M Tris, PH7.5 (for : neutralization) and equal volume of 3 X SDS loading buffer.
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b*******H 发帖数: 830 | 15 THERMO 的Femto 可比普通的ECL灵敏1000倍,平常曝白片的膜换上FEMTO经常是肉眼下
熠熠发光啊,但对有些抗体不行,背景太高。
另外就是世纪太贵。
from
beads
吗?
【在 v***a 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 我也有类似的co-ip不work的困扰。 : 请问你说的: : “Crosslink 1st antibody to beads and elute co-purified protein complex from : beads after washing (instead of boiling beads) to reduce background” : 怎么个crosslink法?我是加了1st Ab overnight再加beads的。 : 还有elute我是用的SDS sample buffer,然后就boil,100 C,10min。boiling beads : 和elute是两个独立的过程呀,为啥elute可以取代boiling?是我理解错你的意思了吗? : 关于ECL plus,我觉得用ECL曝光久一点,貌似也跟plus一样的效果,而且plus还会令 : blot颜色深浅不均一,不知道你会有这个问题吗?如果有,怎么解决比较好? : 谢谢回答。
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