c********r
发帖数: 189 | 1 用的是Bethyl的抗体，做co-IP,研究的蛋白A主要bind到chromatin上。
用bethyl的方法做的cell lysis, 然后co-IP，work。但是我发现，他们的lysis
buffer太mild，A蛋白主要还是留着pellet中。所以IP下来的只是一小部分，可能是
在cytoplasmic里的A蛋白
于是同时用nuclear extract来做co-IP，input里有很多A蛋白，但是co-IP不work，A蛋白没有pull down下来
所以问题是，为啥同一个antibody，可以pull down cyto fraction里的蛋白A,而不可以pull down nuclear内的蛋白A？
我的解释是，在nuclear extract里，A跟很多蛋白结合，于是antigen被mask了。。。
不知道还有没有别的解释
谢谢各位了 |
w******y
发帖数: 2504 | 2 As you suggested, maybe because of co-factors. Or, the A protein is
trunkated when it is moving into the nucleus. |
s*********t
发帖数: 600 | 3 盐浓度？nuclear extract怎么提取的 |
g*********5
发帖数: 2533 | 4 顺求细胞核提取protocol for Coip
【在 s*********t 的大作中提到】
: 盐浓度？nuclear extract怎么提取的
|
l**********1
发帖数: 5204 | 5 please go to
//www.proteinguru.com/protocols/IP%20guide2.pdf
or
//webdoc.nyumc.org/nyumc/files/parasitology/u6/UF_2002_MM.pdf
more:
//webdoc.nyumc.org/nyumc/files/sun-lab/attachments/CPCB.ch03.Cell%
20Fractionation.pdf
【在 g*********5 的大作中提到】
: 顺求细胞核提取protocol for Coip
|
c********r
发帖数: 189 | 6 用的是Bethyl的抗体，做co-IP,研究的蛋白A主要bind到chromatin上。
用bethyl的方法做的cell lysis, 然后co-IP，work。但是我发现，他们的lysis
buffer太mild，A蛋白主要还是留着pellet中。所以IP下来的只是一小部分，可能是
在cytoplasmic里的A蛋白
于是同时用nuclear extract来做co-IP，input里有很多A蛋白，但是co-IP不work，A蛋白没有pull down下来
所以问题是，为啥同一个antibody，可以pull down cyto fraction里的蛋白A,而不可以pull down nuclear内的蛋白A？
我的解释是，在nuclear extract里，A跟很多蛋白结合，于是antigen被mask了。。。
不知道还有没有别的解释
谢谢各位了 |
w******y
发帖数: 2504 | 7 As you suggested, maybe because of co-factors. Or, the A protein is
trunkated when it is moving into the nucleus. |
s*********t
发帖数: 600 | 8 盐浓度？nuclear extract怎么提取的 |
g*********5
发帖数: 2533 | 9 顺求细胞核提取protocol for Coip
【在 s*********t 的大作中提到】
: 盐浓度？nuclear extract怎么提取的
|
l**********1
发帖数: 5204 | 10 please go to
//www.proteinguru.com/protocols/IP%20guide2.pdf
or
//webdoc.nyumc.org/nyumc/files/parasitology/u6/UF_2002_MM.pdf
more:
//webdoc.nyumc.org/nyumc/files/sun-lab/attachments/CPCB.ch03.Cell%
20Fractionation.pdf
【在 g*********5 的大作中提到】
: 顺求细胞核提取protocol for Coip
|
|
|
c********r
发帖数: 189 | 11
It's not truncated because it was detected in the input
【在 w******y 的大作中提到】
: As you suggested, maybe because of co-factors. Or, the A protein is
: trunkated when it is moving into the nucleus.
|
c********r
发帖数: 189 | 12
salt concentration is the ~300mM
cells were treated with hypotonic buffer with Triton 100.nuclei is separated
by low speed centrifugation. NH4SO4 were added to a final concentration of
300mM to precipitate the chromatin; then centrifuge to separate nuclear
protein from chromatin, save the supernatant.
add 1:1 3M NH4SO4 to precipitate the protein, and then dissolve protein
pellet with your IP buffer.
【在 s*********t 的大作中提到】
: 盐浓度？nuclear extract怎么提取的
|
c********r
发帖数: 189 | 13
cells were treated with hypotonic buffer with Triton 100.nuclei is separated
by low speed centrifugation. NH4SO4 were added to a final concentration of
300mM to precipitate the chromatin; then centrifuge to separate nuclear
protein from chromatin, save the supernatant.
add 1:1 3M NH4SO4 to precipitate the protein, and then dissolve protein
pellet with your IP buffer.
【在 g*********5 的大作中提到】
: 顺求细胞核提取protocol for Coip
|
s*********t
发帖数: 600 | 14 为何最后一步要先用NH4SO4沉淀，再用IP buffer 溶解？这么剧烈的条件对于蛋白不好
吧？
直接透析到IP buffer甚至直接稀释，如何？
separated
【在 c********r 的大作中提到】
:
: cells were treated with hypotonic buffer with Triton 100.nuclei is separated
: by low speed centrifugation. NH4SO4 were added to a final concentration of
: 300mM to precipitate the chromatin; then centrifuge to separate nuclear
: protein from chromatin, save the supernatant.
: add 1:1 3M NH4SO4 to precipitate the protein, and then dissolve protein
: pellet with your IP buffer.
|
g*********5
发帖数: 2533 | 15 thanks for your help. can I just use some kits for the experiment? |
r*******y
发帖数: 48 | |
