b*******n
发帖数: 605 | 1 Hi folks,
I am doing a CO-IP for an endogenous nuclear protein. I have tried several
protocols with acid wash but have not
been successful even once so far. Can anybody kindly share a protocol with
me if it works for you? Or can
anybody give suggestions for trouble shooting? Thank you! |
w********r
发帖数: 1431 | 2 如果是很难做的endogenous CO-IP,不妨先用DSP crosslink一下再做，
虽然很多人鄙视这种corsslink后做出来的CO-IP结果，但只要做好各种control，我觉
得其实也还可以。 |
w********r
发帖数: 1431 | 3 1.wash cell twice with PBS
2. incubate with DSP/DMSO/PBS solution for 20min at RT (freshly!!! prepared
DSP,2.5mg DSP-->480ul DMSO-->12ml PBS, final 200ug/ml DSP)
3. wash 2XPBS
4.incubate 5min with 50mM Glycine/PBS
5. wash with PBS
6. harvest cell in RIPA buffer with PIM, w/o DTT!!!， followed by CO-IP. Wash stringently with RIPA before WB.
Good luck!
【在 b*******n 的大作中提到】
: Hi folks,
: I am doing a CO-IP for an endogenous nuclear protein. I have tried several
: protocols with acid wash but have not
: been successful even once so far. Can anybody kindly share a protocol with
: me if it works for you? Or can
: anybody give suggestions for trouble shooting? Thank you!
|
c*********r
发帖数: 1312 | 4 推荐pierce的这个kit：http://www.piercenet.com/products/browse.cfm?fldID=01010345
也要cross link，对抗体量要求比较高。目前用着不错，但还没有做mass spec验证。
。。 |
b*******n
发帖数: 605 | 5 Thank you a lot, guys!!! |
b*******n
发帖数: 605 | 6 Do you know if this kit is also good for CO-IP?
Thank you.
【在 c*********r 的大作中提到】
: 推荐pierce的这个kit：http://www.piercenet.com/products/browse.cfm?fldID=01010345
: 也要cross link，对抗体量要求比较高。目前用着不错，但还没有做mass spec验证。
: 。。
|
j*****a
发帖数: 658 | 7 I read through the link and the advantage of this product is basically they
eliminate the contamination of antibody fragments in the immunoblot
following IP. It will give you less non-specfic background of IG heavy and
light chain. It has nothing to do with your problem which is no band
detected. Also one biggest disadvantage of this product is that, when IP
antibody is immobilized to the beads, there is possibility that it gets de-
activated somehow. Bascially you tend to have negative results.
【在 b*******n 的大作中提到】
: Do you know if this kit is also good for CO-IP?
: Thank you.
|
b*******n
发帖数: 605 | 8 Thanks!
Yes, that is exactly why I am asking this question.
they
【在 j*****a 的大作中提到】
: I read through the link and the advantage of this product is basically they
: eliminate the contamination of antibody fragments in the immunoblot
: following IP. It will give you less non-specfic background of IG heavy and
: light chain. It has nothing to do with your problem which is no band
: detected. Also one biggest disadvantage of this product is that, when IP
: antibody is immobilized to the beads, there is possibility that it gets de-
: activated somehow. Bascially you tend to have negative results.
|
c*********r
发帖数: 1312 | 9 我目前用着还好，除了目的蛋白，还有至少三四个条带，目前正在分析中。。。
【在 b*******n 的大作中提到】
: Do you know if this kit is also good for CO-IP?
: Thank you.
|
c*********r
发帖数: 1312 | 10 能分享一下为什么很多人鄙视crosslink做出的co-IP吗？什么样的control比较好。我
用两种control：1. 没加抗体，只用Protain A/G的resin；2. 用同样动物（兔子）的
IgG，但是是不针对我的体系里任何蛋白的抗体，也就是non-relevant antibody。
【在 w********r 的大作中提到】
: 如果是很难做的endogenous CO-IP,不妨先用DSP crosslink一下再做，
: 虽然很多人鄙视这种corsslink后做出来的CO-IP结果，但只要做好各种control，我觉
: 得其实也还可以。
|
|
|
c*********r
发帖数: 1312 | 11 “there is possibility that it gets de-activated somehow”
我也觉得是有这个可能，不然这个kit不会要求10 ug左右的抗体。
以前做不用crosslink的做，好像只要1-2 ug就够了。。。
they
【在 j*****a 的大作中提到】
: I read through the link and the advantage of this product is basically they
: eliminate the contamination of antibody fragments in the immunoblot
: following IP. It will give you less non-specfic background of IG heavy and
: light chain. It has nothing to do with your problem which is no band
: detected. Also one biggest disadvantage of this product is that, when IP
: antibody is immobilized to the beads, there is possibility that it gets de-
: activated somehow. Bascially you tend to have negative results.
|
c*********r
发帖数: 1312 | 12 还有一个问题，我是用银染检测的，你是用什么方法染胶？灵敏度有多高？这个也很关
键。^_^ |
b*******n
发帖数: 605 | 13 Really? I am actually doing the exact same experiment as you are.
Before, I used acid wash to get rid of non-specifc and IgG, but the silver
staining still runs terrible, which
makes the MS impossible. If you get only 3 or 4 bands, it is really awesome
!
Do you work on infected cultured-cells, or tissue? I am using tissue for my
experiment.
【在 c*********r 的大作中提到】
: 我目前用着还好，除了目的蛋白，还有至少三四个条带，目前正在分析中。。。
|
b*******n
发帖数: 605 | 14 I have a candidate protein, so I used it as positive control. I did western
blots to detect it, which should be
more sensitive than silver staining...
【在 c*********r 的大作中提到】
: 还有一个问题，我是用银染检测的，你是用什么方法染胶？灵敏度有多高？这个也很关
: 键。^_^
|
j*****a
发帖数: 658 | 15 what lysis buffer did you use? I am wondering if RIPA buffer/denature buffer
is okay for IP......
Do you know anything about this? THanks!
western
【在 b*******n 的大作中提到】
: I have a candidate protein, so I used it as positive control. I did western
: blots to detect it, which should be
: more sensitive than silver staining...
|
s******s
发帖数: 13035 | 16 buffer都要试的，背景比较强的就上1% Triton-X100, 甚至0.1%SDS，
比较弱的只能0.1% Tween啥的了
buffer
【在 j*****a 的大作中提到】
: what lysis buffer did you use? I am wondering if RIPA buffer/denature buffer
: is okay for IP......
: Do you know anything about this? THanks!
:
: western
|
j*****a
发帖数: 658 | 17 I am not sure if I understand you well...I thought the difference of non-
ionic (NP40, Triton X100) and ionic (SDS, sodium deoxycholate) is non-ionic
lysis buffer doesn't denature protein. Some of the antibody that recognizes
only non-denature antigen (original state, you might want to say) so that
you can't get good results if you use denature lysis buffer (SDS) with these
antibodies. In anothe word, non-denature lysis buffer is more safe. Am I
wrong?
So I am confused with what you said about "high background uses triton or
SDS, low background is tween"...what does it mean? sorry, i am new to this
technique...
Thanks
【在 s******s 的大作中提到】
: buffer都要试的，背景比较强的就上1% Triton-X100, 甚至0.1%SDS，
: 比较弱的只能0.1% Tween啥的了
:
: buffer
|
c*********r
发帖数: 1312 | 18 我没有用过acid wash，我都是按照那个kit的protocol来的，用lysis buffer或者TBS
wash。其实我的银染的条带也目前也没有用western检测到，同实验室的博后检测到过
，但是需要蛋白上样量是我银染的五倍才勉强看到一条带。我们是用Li-Cor Odyssey的
Infrared western，理论上和银染灵敏度相当，但我持怀疑态度。上一个图片，背景比
较大，左边的是对照，右边的是IP，银染，按照kit的protocol的第二次的elution，10
ul上样量。
awesome
my
【在 b*******n 的大作中提到】
: Really? I am actually doing the exact same experiment as you are.
: Before, I used acid wash to get rid of non-specifc and IgG, but the silver
: staining still runs terrible, which
: makes the MS impossible. If you get only 3 or 4 bands, it is really awesome
: !
: Do you work on infected cultured-cells, or tissue? I am using tissue for my
: experiment.
|
c*********r
发帖数: 1312 | 19 我用的是sea urchin eggs，所有蛋白都是纯天然的。^_^ 不是转染、过表达的。一个
IP用2mg到8mg total protein。起始细胞重量大概是总蛋白的10倍左右。
awesome
my
【在 b*******n 的大作中提到】
: Really? I am actually doing the exact same experiment as you are.
: Before, I used acid wash to get rid of non-specifc and IgG, but the silver
: staining still runs terrible, which
: makes the MS impossible. If you get only 3 or 4 bands, it is really awesome
: !
: Do you work on infected cultured-cells, or tissue? I am using tissue for my
: experiment.
|
j*****a
发帖数: 658 | 20 which one is your target protein band? biggest band?
TBS
10
【在 c*********r 的大作中提到】
: 我没有用过acid wash，我都是按照那个kit的protocol来的，用lysis buffer或者TBS
: wash。其实我的银染的条带也目前也没有用western检测到，同实验室的博后检测到过
: ，但是需要蛋白上样量是我银染的五倍才勉强看到一条带。我们是用Li-Cor Odyssey的
: Infrared western，理论上和银染灵敏度相当，但我持怀疑态度。上一个图片，背景比
: 较大，左边的是对照，右边的是IP，银染，按照kit的protocol的第二次的elution，10
: ul上样量。
:
: awesome
: my
|
|
|
c*********r
发帖数: 1312 | 21 红箭头所示。其实这个条带的大小和预计的以及以前western的结果一致，我还需要再
做一次IP的western来确认。
【在 j*****a 的大作中提到】
: which one is your target protein band? biggest band?
:
: TBS
: 10
|
b*******n
发帖数: 605 | 22 pretty!
However, the heavy band in the middle and at the bottom still look like
heavy chain and light chain. Have you
checked their MW and are they ? If yes, isn't that the kits suppose to get rid of the heavy
and light chain stuff?
TBS
10
【在 c*********r 的大作中提到】
: 我没有用过acid wash，我都是按照那个kit的protocol来的，用lysis buffer或者TBS
: wash。其实我的银染的条带也目前也没有用western检测到，同实验室的博后检测到过
: ，但是需要蛋白上样量是我银染的五倍才勉强看到一条带。我们是用Li-Cor Odyssey的
: Infrared western，理论上和银染灵敏度相当，但我持怀疑态度。上一个图片，背景比
: 较大，左边的是对照，右边的是IP，银染，按照kit的protocol的第二次的elution，10
: ul上样量。
:
: awesome
: my
|
s******s
发帖数: 13035 | 23 嗯，我没想过这个denature的问题。
对我来说，non-ionic对lipid作用比较强，对蛋白－蛋白作用弱，所以能去除比较弱
的蛋白相互作用。ionic的分离蛋白作用强点，0.1%SDS还是很稀，不至于蛋白全变性，
能够去掉中等的相互作用，并且溶解一下不好溶的蛋白。
ionic
recognizes
these
【在 j*****a 的大作中提到】
: I am not sure if I understand you well...I thought the difference of non-
: ionic (NP40, Triton X100) and ionic (SDS, sodium deoxycholate) is non-ionic
: lysis buffer doesn't denature protein. Some of the antibody that recognizes
: only non-denature antigen (original state, you might want to say) so that
: you can't get good results if you use denature lysis buffer (SDS) with these
: antibodies. In anothe word, non-denature lysis buffer is more safe. Am I
: wrong?
: So I am confused with what you said about "high background uses triton or
: SDS, low background is tween"...what does it mean? sorry, i am new to this
: technique...
|
w********r
发帖数: 1431 | 24 真的猛男只关心那些巨强无比怎么洗也洗不散的强binding，
对需要corsslink才能做出来的当然就不屑了
【在 c*********r 的大作中提到】
: 能分享一下为什么很多人鄙视crosslink做出的co-IP吗？什么样的control比较好。我
: 用两种control：1. 没加抗体，只用Protain A/G的resin；2. 用同样动物（兔子）的
: IgG，但是是不针对我的体系里任何蛋白的抗体，也就是non-relevant antibody。
|
b*******n
发帖数: 605 | 25 haha, I love this!
【在 w********r 的大作中提到】
: 真的猛男只关心那些巨强无比怎么洗也洗不散的强binding，
: 对需要corsslink才能做出来的当然就不屑了
|
s******s
发帖数: 13035 | 26 上次谁说来着，真正的co-ip就是pull down下来，然后用最gentle的条件
稍微洗一次，马上趁热呼上样来着
【在 w********r 的大作中提到】
: 真的猛男只关心那些巨强无比怎么洗也洗不散的强binding，
: 对需要corsslink才能做出来的当然就不屑了
|
c*********r
发帖数: 1312 | 27 You are right. 我觉得中间的也是heavy chain。但是我觉得原因是这样的，抗体放久
了有些聚集，crosslink时大部分交联了，部分还是和其它的抗体结合在一起，然后被
我洗脱下来了。我猜是这个原因是因为重复用过几次的Resin再用的话就没有这个现象
了。
rid of the heavy
【在 b*******n 的大作中提到】
: pretty!
: However, the heavy band in the middle and at the bottom still look like
: heavy chain and light chain. Have you
: checked their MW and are they ? If yes, isn't that the kits suppose to get rid of the heavy
: and light chain stuff?
:
: TBS
: 10
|
c*********r
发帖数: 1312 | 28 我想我明白了你说的crosslink和我说的crosslink不是一个东东。。。
我说的是把抗体和beads上的protein A/G交联起来，你可能是指把IP complex里的所有
protein crosslink吧。
不知道我说的对不对。^_^
【在 w********r 的大作中提到】
: 真的猛男只关心那些巨强无比怎么洗也洗不散的强binding，
: 对需要corsslink才能做出来的当然就不屑了
|
w********r
发帖数: 1431 | 29 是的，你得到了它：）
LZ问题是CO-IP拖不下东西来要怎么优化。
那么用DSP把细胞内的蛋白连一下，一些比较弱或者transient的binding可以被固定住
，比较容易检测一点，也不用费劲去优化各种buffer的条件——虽然很多人看不上这种
CO-IP.
至于你们讨论的抗体和beads crosslink 可以消除IgG chains，但同时会导致抗体结合的效率更加低。
【在 c*********r 的大作中提到】
: 我想我明白了你说的crosslink和我说的crosslink不是一个东东。。。
: 我说的是把抗体和beads上的protein A/G交联起来，你可能是指把IP complex里的所有
: protein crosslink吧。
: 不知道我说的对不对。^_^
|
