m******5
发帖数: 1383 | 1 目的蛋白是个transcription factor，表达很低。
看了不少Chip protocol，似乎stringency都很高，貌似都是给 histon之类表达量高的
蛋白用的，有同修接触过类似情况么? |
d****7
发帖数: 109 | |
d****i
发帖数: 2346 | 3 顺路问一下，这种表达量很低的TF能不能转染进去让表达高一些，然后做CHIP？ |
t**l
发帖数: 109 | 4 可以，但是那样就不是physiological 的结果了。不过你转进去的protein TF要是带
TAG，就用TAG做CHIP. 省了抗体的钱了。 注意要一个non-sepcific recruitment
control. TF overexpression的话哪里都bind,你的negative control region可能
background也会很高。要是做转染，在转一个DNA binding domain amino acid 突变体
，然后再做CHIP,这样就能控制你的结果。 |
m******5
发帖数: 1383 | 5 Non specific recruitment comtrol怎么做？ 另外如果做sequencing的话这样出来的
结果不是很可怕
【在 t**l 的大作中提到】
: 可以，但是那样就不是physiological 的结果了。不过你转进去的protein TF要是带
: TAG，就用TAG做CHIP. 省了抗体的钱了。 注意要一个non-sepcific recruitment
: control. TF overexpression的话哪里都bind,你的negative control region可能
: background也会很高。要是做转染，在转一个DNA binding domain amino acid 突变体
: ，然后再做CHIP,这样就能控制你的结果。
|
m******5
发帖数: 1383 | 6 请教一下，在没有per existing known binding motif的情况下，怎么做quantity
control for chip seq?
【在 d****7 的大作中提到】
: 其实还是抗体最重要
: rick young的lab protocol很好，我一直用，做了20多个TF ChIPseq，抗体行的话没有
: 任何问题
: http://www.nature.com/nprot/journal/v1/n2/abs/nprot.2006.98.htm
: http://www.ncbi.nlm.nih.gov/pubmed/19275939
|
y*********u
发帖数: 183 | 7 What I could think of: you can run a westernblot using CHIP samples before
protenase K treatment(1/10-1/20 depend on the expression level of your
protein of interest). If you pulled down a lot of your protein of interest,
and if your procedure is clean, I see no reason the CHIP-Seq wouldn't work
if your protein is indeed a DNA binding protein.
In worst case scenario you should still be able to get lots of strong peaks
which is particularly useful to you since your problem is that you didn't
have a preexisting binding site to start with, now you can have it.
【在 m******5 的大作中提到】
: 请教一下，在没有per existing known binding motif的情况下，怎么做quantity
: control for chip seq?
|
m******5
发帖数: 1383 | 8 thank you very much!!!!!
【在 y*********u 的大作中提到】
: What I could think of: you can run a westernblot using CHIP samples before
: protenase K treatment(1/10-1/20 depend on the expression level of your
: protein of interest). If you pulled down a lot of your protein of interest,
: and if your procedure is clean, I see no reason the CHIP-Seq wouldn't work
: if your protein is indeed a DNA binding protein.
: In worst case scenario you should still be able to get lots of strong peaks
: which is particularly useful to you since your problem is that you didn't
: have a preexisting binding site to start with, now you can have it.
|
y*********1
发帖数: 46 | 9 You can test to see if your TF binds its own promoter. Quite often, a TF
will bind to its own promoter. |
m******5
发帖数: 1383 | 10 Thanks! Do you have a reference for that?
I was trying to look for it.
【在 y*********1 的大作中提到】
: You can test to see if your TF binds its own promoter. Quite often, a TF
: will bind to its own promoter.
|
y*********1
发帖数: 46 | 11 Search Pubmed with "ChIP-Seq" and "autoregulation" |
