g*********3
发帖数: 177 | 1 可能要做p300的ChIP-seq,大家推荐下antibody。用mouse tissue做，实验室里已经试
了santa cruz的SC-585，对人的细胞系还可以，对老鼠好像不太行。
另外对enhancer的ChIP-seq除了p300还没有别的可以互补的，比如CBP啥的。
谢谢各位了先。 |
m******5
发帖数: 1383 | |
g*********3
发帖数: 177 | 3 Thanks for your answer.
This is the antibody we have tried before. It doesn't work very well in
mouse although it works in human cell line.
Anyway, thanks.
【在 m******5 的大作中提到】
: http://www.nature.com/nature/journal/v457/n7231/full/nature0773
|
m******5
发帖数: 1383 | 4 Sorry for being not so helpful………… but the experiment in the paper was
done in mice right?
【在 g*********3 的大作中提到】
: Thanks for your answer.
: This is the antibody we have tried before. It doesn't work very well in
: mouse although it works in human cell line.
: Anyway, thanks.
|
g*********3
发帖数: 177 | 5 Yes you are right. This antibody has been used in several groups to do ChiP-
SEQ on p300. I don't understand why it doesn't work very well in our mouse
cell line.
Thanks.
【在 m******5 的大作中提到】
: Sorry for being not so helpful………… but the experiment in the paper was
: done in mice right?
|
m******5
发帖数: 1383 | 6 oh, that sucks, did you try to purchase the same antibody from other
companies? I was under the impression that the paper I attached actually
gave very specific result at the end denoting that the element they
identified can actually function in a developmental regulated manner.
also I strongly suggest you to change the title of your post to a more
specific one
ChiP-
【在 g*********3 的大作中提到】
: Yes you are right. This antibody has been used in several groups to do ChiP-
: SEQ on p300. I don't understand why it doesn't work very well in our mouse
: cell line.
: Thanks.
|
g*********3
发帖数: 177 | 7 嗯，你说的没错。文章里确实有很specific的东西.
但是很奇怪，这个抗体在人的细胞系中工作很好，在mouse liver tissue中做完ChIP之
后library基本看不到DNA...
还是继续求P300 antibody （在mouse liver tissue做ChIP-seq的）
谢谢你啦～
也谢谢各位.
【在 m******5 的大作中提到】
: oh, that sucks, did you try to purchase the same antibody from other
: companies? I was under the impression that the paper I attached actually
: gave very specific result at the end denoting that the element they
: identified can actually function in a developmental regulated manner.
: also I strongly suggest you to change the title of your post to a more
: specific one
:
: ChiP-
|
E*********s
发帖数: 93 | 8 The P300 antibody used in that Nature paper does work, however, you need to get the exact lot, which is very hard now. Also I found different tissues have diffferent enrichment of P300, and some tissues don't have any P300 signal in ChIP-seq experiments, and I remember liver is one of them. |
g*********3
发帖数: 177 | 9 Hi,
Thanks for your answer.
It is really ridiculous...
It is reasonable that different tissues have different enrichment of P300
since it may indicate there exist some other coactivators besides P300.
In this case, do you think it is because the P300 antibody doesn't work in
liver tissue for some unknown reasons or because P300 is not the main player
...(Have you done the western blot on nuclear extract against P300 in the
tissue that doesn't fail in P300 ChIP?)
Thank you very much.
to get the exact lot, which is very hard now. Also I found different tissues
have diffferent enrichment of P300, and some tissues don't have any P300
signal in ChIP-seq experiments, and I remember liver is one of them.
【在 E*********s 的大作中提到】
: The P300 antibody used in that Nature paper does work, however, you need to get the exact lot, which is very hard now. Also I found different tissues have diffferent enrichment of P300, and some tissues don't have any P300 signal in ChIP-seq experiments, and I remember liver is one of them.
|
E*********s
发帖数: 93 | 10 Doing western is a very good suggestion. I didn't do the western yet. Maybe you can try that first and tell me
know. It is reported in some cancer cells, P300 C-terminus is truncated. Don
't if that is the case for different normal tissues. |
E*********s
发帖数: 93 | 11 You can also use distal H3K27Ac sites to identify enhancers. Also present of
H3K4me1 and absence of H3K4me3 at distal sites can also be used to identify
enhancer. |
g*********3
发帖数: 177 | 12 谢谢你的回复.
新年快乐～
H3K27AC是我们的一个选择，但是有paper说抗体特异性不是很好～
关于p300的抗体我给你发了私信.
谢谢了.
of
identify
【在 E*********s 的大作中提到】
: You can also use distal H3K27Ac sites to identify enhancers. Also present of
: H3K4me1 and absence of H3K4me3 at distal sites can also be used to identify
: enhancer.
|
