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发信人: gardenia (缘了，就是完), 信区: Biology
标 题: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Thu Oct 3 17:43:18 2002), 站内信件
我现在需要做一个蛋白的western blot
因为样品中含量很小，我想先做IP然后再Western
问题是我只有一种这个蛋白的antibody，因此做western的时候这个Ab
总是能够显示出来，而且其大小正好和我要做的蛋白一致。
我想问的问题是，有没有办法在做完免疫沉降之后，有办法能将抗体从我的样品里面去掉？
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.61.]
发信人: bongbongcat (cutie), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Thu Oct 3 19:46:12 2002), 转信
cross link your ab to the beads before adding it to the cell lysate. this
way, it will not be released from the beads with boiling in sample buffer.
good luck
【 在 gardenia (缘了，就是完) 的大作中提到: 】
: 我现在需要做一个蛋白的western blot
: 因为样品中含量很小，我想先做IP然后再Western
: 问题是我只有一种这个蛋白的antibody，因此做western的时候这个Ab
: 总是能够显示出来，而且其大小正好和我要做的蛋白一致。
: 我想问的问题是，有没有办法在做完免疫沉降之后，有办法能将抗体从我的样品里面去掉？
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 68.81.]
发信人: forrestgump (sleepless in Houston), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Thu Oct 3 20:42:21 2002) WWW-POST
it will reduce the amount of contaminating Ab, but will not completely remove
it because of the leakness of the crosslinking (Pierce has better x-linking
kit?). so you will have to find another way to detect it.
【 在 bongbongcat (cutie) 的大作中提到: 】
: cross link your ab to the beads before adding it to the cell lysate. this
: way, it will not be released from the beads with boiling in sample buffer.
: good luck
:
: 【 在 gardenia (缘了，就是完) 的大作中提到: 】
: : 我现在需要做一个蛋白的western blot
: : 因为样品中含量很小，我想先做IP然后再Western
: : 问题是我只有一种这个蛋白的antibody，因此做western的时候这个Ab
: : 总是能够显示出来，而且其大小正好和我要做的蛋白一致。
: : 我想问的问题是，有没有办法在做完免疫沉降之后，有办法能将抗体从我的样品里面
去掉？
:
:
--
LIfe is like a box of chocolate.
You never know what is going to be the next.
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.249.]
发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Thu Oct 3 21:43:12 2002), 站内信件
your problem is not western. the key problem is that the
amount of the endogenous protein that you are interested
is so small that you could hardly detect it by common
immunoblot. you may try to use some hypersensitive film
for visulization, or enrich the protein as bongbongcat has
suggested. btw, i would rather do in situ hyb for RNA. how
much would like to know? is the western a necessity for
manuscript? i have seen many papers with no expression profile
of endogenous protein. and what they usually do is to over
express the protein in some cell lines that have very low
amount of such protein. but technically speaking, i would not
quite favor such method since it overloads the cell system with
enormous amount of protein. so could you specify your question
since for different proposal you have to use diff. approaches.
【 在 bongbongcat (cutie) 的大作中提到: 】
: cross link your ab to the beads before adding it to the cell lysate. this
: way, it will not be released from the beads with boiling in sample buffer.
: good luck
: 【 在 gardenia (缘了，就是完) 的大作中提到: 】
: : 我现在需要做一个蛋白的western blot
: : 因为样品中含量很小，我想先做IP然后再Western
: : 问题是我只有一种这个蛋白的antibody，因此做western的时候这个Ab
: : 总是能够显示出来，而且其大小正好和我要做的蛋白一致。
: : 我想问的问题是，有没有办法在做完免疫沉降之后，有办法能将抗体从我的样品里面去掉？
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 146.186.]
发信人: krait (我的寂寞是一条蛇), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Fri Oct 4 02:29:21 2002) WWW-POST
Pierce kit sieze-X.
【 在 xcosmos (hi) 的大作中提到: 】
: what do you mean by "cross link"?
: how to do that?
: thanks
: 【 在 bongbongcat (cutie) 的大作中提到: 】
: : cross link your ab to the beads before adding it to the cell lysate. this
: : way, it will not be released from the beads with boiling in sample buffer.
: : good luck
: :
: : 【 在 gardenia (缘了，就是完) 的大作中提到: 】
: : : 我现在需要做一个蛋白的western blot
: : : 因为样品中含量很小，我想先做IP然后再Western
: : : 问题是我只有一种这个蛋白的antibody，因此做western的时候这个Ab
: : : 总是能够显示出来，而且其大小正好和我要做的蛋白一致。
: : : 我想问的问题是，有没有办法在做完免疫沉降之后，有办法能将抗体从我的样品里
面
: 去掉？
: :
: :
:
:
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 65.71.]
发信人: gardenia (缘了，就是完), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Fri Oct 4 10:08:24 2002), 站内信件
the problem is my protein is around 55kDa
the same as heavy chain of primary Ab
and i always see a strong band there, i know it is the Ab,
but i don't know whether my protein is also there
【 在 Marble (小石头哥哥) 的大作中提到: 】
: your problem is not western. the key problem is that the
: amount of the endogenous protein that you are interested
: is so small that you could hardly detect it by common
: immunoblot. you may try to use some hypersensitive film
: for visulization, or enrich the protein as bongbongcat has
: suggested. btw, i would rather do in situ hyb for RNA. how
: much would like to know? is the western a necessity for
: manuscript? i have seen many papers with no expression profile
: of endogenous protein. and what they usually do is to over
: express the protein in some cell lines that have very low
: amount of such protein. but technically speaking, i would not
: quite favor such method since it overloads the cell system with
: enormous amount of protein. so could you specify your question
: since for different proposal you have to use diff. approaches.
: 【 在 bongbongcat (cutie) 的大作中提到: 】
: : cross link your ab to the beads before adding it to the cell lysate. this
: : way, it will not be released from the beads with boiling in sample buffer.
: : good luck
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 170.140.]
发信人: sunnyday (飞鸟), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Fri Oct 4 12:24:26 2002) WWW-POST
Just like other gus have already said:
1. Buy another antibody from another specie. (I highly recommend this way)
2. crosslink your antibody to gel particles (CNBr activated Sepharose),
use the gel to pull down your protein,then use high salt(eg., 8M urea) to
seperate your protein.
3. crosslink your antibody to your protein by EDC-NHS method, then your
target will have a higher molecular weight. However, the result can turn out
to be hard to interpreted since the antibody would also bind something else
although with lower affinity.
【 在 gardenia (缘了，就是完) 的大作中提到: 】
: the problem is my protein is around 55kDa
: the same as heavy chain of primary Ab
: and i always see a strong band there, i know it is the Ab,
: but i don't know whether my protein is also there
: 【 在 Marble (小石头哥哥) 的大作中提到: 】
: : your problem is not western. the key problem is that the
: : amount of the endogenous protein that you are interested
: : is so small that you could hardly detect it by common
: : immunoblot. you may try to use some hypersensitive film
: : for visulization, or enrich the protein as bongbongcat has
: : suggested. btw, i would rather do in situ hyb for RNA. how
: : much would like to know? is the western a necessity for
: : manuscript? i have seen many papers with no expression profile
: : of endogenous protein. and what they usually do is to over
: : express the protein in some cell lines that have very low
: : amount of such protein. but technically speaking, i would not
: : quite favor such method since it overloads the cell system with
: : enormous amount of protein. so could you specify your question
: : since for different proposal you have to use diff. approaches.
: : 【 在 bongbongcat (cutie) 的大作中提到: 】
: : : cross link your ab to the beads before adding it to the cell lysate.
this
: : : way, it will not be released from the beads with boiling in sample
buffer.
: : : good luck
:
:
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 159.178.]
发信人: stonecold (deja+vu), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Fri Oct 4 20:54:24 2002), 站内信件
strange
if you can IP the protein,
what's the propose to do western?
it's like "you've already proved this flower is red, and then you go ahead
to prove it's not yellow"/
【 在 gardenia (缘了，就是完) 的大作中提到: 】
: 我现在需要做一个蛋白的western blot
: 因为样品中含量很小，我想先做IP然后再Western
: 问题是我只有一种这个蛋白的antibody，因此做western的时候这个Ab
: 总是能够显示出来，而且其大小正好和我要做的蛋白一致。
: 我想问的问题是，有没有办法在做完免疫沉降之后，有办法能将抗体从我的样品里面去掉？
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.218.]
发信人: stonecold (deja+vu), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Fri Oct 4 21:05:09 2002), 站内信件
western can be a good way to quantitate protein concentration?
you gotta be kidding me.
【 在 Eddy (打哪儿指哪儿) 的大作中提到: 】
: what if he wants to quntitate this protein's concentration in different samp
: les?
: 【 在 stonecold (deja+vu) 的大作中提到: 】
: : strange
: : if you can IP the protein,
: : what's the propose to do western?
: : it's like "you've already proved this flower is red, and then you go ahead
: : to prove it's not yellow"/
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.218.]
发信人: gardenia (缘了，就是完), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Fri Oct 4 21:35:22 2002), 站内信件
i can't prove I can IP the protein, either
i just try to IP and western it, got it?
because after IP, my protein still will be migrating with heavy chain and
shows up in the same band in SDS-PAGE
【 在 stonecold (deja+vu) 的大作中提到: 】
: strange
: if you can IP the protein,
: what's the propose to do western?
: it's like "you've already proved this flower is red, and then you go ahead
: to prove it's not yellow"/
: 【 在 gardenia (缘了，就是完) 的大作中提到: 】
: : 我现在需要做一个蛋白的western blot
: : 因为样品中含量很小，我想先做IP然后再Western
: : 问题是我只有一种这个蛋白的antibody，因此做western的时候这个Ab
: : 总是能够显示出来，而且其大小正好和我要做的蛋白一致。
: : 我想问的问题是，有没有办法在做完免疫沉降之后，有办法能将抗体从我的样品里面去掉？
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 170.140.]
发信人: gardenia (缘了，就是完), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Fri Oct 4 21:36:36 2002), 站内信件
yeah, i know the 1 is a good solution
the problem is no antibody is available for that protein other than
from rabbit
【 在 sunnyday (飞鸟) 的大作中提到: 】
: Just like other gus have already said:
: 1. Buy another antibody from another specie. (I highly recommend this way)
: 2. crosslink your antibody to gel particles (CNBr activated Sepharose),
: use the gel to pull down your protein,then use high salt(eg., 8M urea) to
: seperate your protein.
: 3. crosslink your antibody to your protein by EDC-NHS method, then your
: target will have a higher molecular weight. However, the result can turn out
: to be hard to interpreted since the antibody would also bind something else
: although with lower affinity.
: 【 在 gardenia (缘了，就是完) 的大作中提到: 】
: : the problem is my protein is around 55kDa
: : the same as heavy chain of primary Ab
: : and i always see a strong band there, i know it is the Ab,
: : but i don't know whether my protein is also there
: this
: buffer.
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 170.140.]
发信人: sunnyday (飞鸟), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Sat Oct 5 01:41:34 2002) WWW-POST
You gonna be kidding me. Have you check out other company? There are many
companies in this world make antibodies. You don't have to stuck with one.
【 在 gardenia (缘了，就是完) 的大作中提到: 】
: yeah, i know the 1 is a good solution
: the problem is no antibody is available for that protein other than
: from rabbit
: 【 在 sunnyday (飞鸟) 的大作中提到: 】
: : Just like other gus have already said:
: : 1. Buy another antibody from another specie. (I highly recommend this way)
: : 2. crosslink your antibody to gel particles (CNBr activated Sepharose),
: : use the gel to pull down your protein,then use high salt(eg., 8M urea) to
: : seperate your protein.
: : 3. crosslink your antibody to your protein by EDC-NHS method, then your
: : target will have a higher molecular weight. However, the result can turn
out
: : to be hard to interpreted since the antibody would also bind something
else
: : although with lower affinity.
: : 【 在 gardenia (缘了，就是完) 的大作中提到: 】
: : : the problem is my protein is around 55kDa
: : : the same as heavy chain of primary Ab
: : : and i always see a strong band there, i know it is the Ab,
: : : but i don't know whether my protein is also there
: : this
: : buffer.
:
:
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.227.]
发信人: gardenia (缘了，就是完), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Sat Oct 5 12:26:35 2002), 站内信件
i checked every company that i can find,just can't find it
that's not a ot protein and only two or three companies making it
【 在 sunnyday (飞鸟) 的大作中提到: 】
: You gonna be kidding me. Have you check out other company? There are many
: companies in this world make antibodies. You don't have to stuck with one.
: 【 在 gardenia (缘了，就是完) 的大作中提到: 】
: : yeah, i know the 1 is a good solution
: : the problem is no antibody is available for that protein other than
: : from rabbit
: out
: else
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 170.140.]
发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Mon Oct 7 17:50:49 2002), 站内信件
either you label the protein with S35 methionin, or run a
long PAGE (say 6% or even 4%) to fractionate the protein
from the Ab. for endogenous protein, maybe running long
gel works.
【 在 gardenia (缘了，就是完) 的大作中提到: 】
: that's not a bad idea, i may try that
: 【 在 akite (戒棋) 的大作中提到: 】
: : why don't you radio-label the protein?
--
※ 修改:．Marble 于 Oct 7 18:08:16 修改本文．[FROM: 146.186.]
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 146.186.]
发信人: sunnyday (飞鸟), 信区: Biology
标 题: Re: 免疫沉降--寻求帮助
发信站: The unknown SPACE (Mon Oct 7 18:43:59 2002) WWW-POST
I think running another column will dilute the protein again.
Also, about radioactivity labelling, I am not sure how you guys gonna make
it work around this problem...
If you label the IgG for IP, then the exposure will show one band on the
MW of IgG on WB membrane.
If you label IgG for WB, then it will show one band on the MW of Tubby on WB
membrane.
However,either band will have similar position since the MW of IgG or Tubby
is close enough to confuse you.
The same problem wil remain.
I would suggest to concentrate the protein sample by other method other than
IP.
If Gardenia wants, he could crosslink the anti-Tubby on a CNBr-activated
gel, then this gel can be used conveniently as a affinity column.
【 在 Marble (小石头哥哥) 的大作中提到: 】
: either you label the protein with S35 methionin, or run a
: long PAGE (say 6% or even 4%) to fractionate the protein
: from the Ab. for endogenous protein, maybe running long
: gel works.
:
: 【 在 gardenia (缘了，就是完) 的大作中提到: 】
: : that's not a bad idea, i may try that
: : 【 在 akite (戒棋) 的大作中提到: 】
: : : why don't you radio-label the protein?
:
:
:
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 159.178.]
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