CHIP western - Western after CHIP (Sep/20/2007 )

Hi!
I want to do a CHIP and then western blot to see associated proteins.
I want to immunoprecipitate from a cell line using antibody against my favorite protein , then once i get a chromatin associated with other proteins in complex i want to do a western to see what other proteins are present. Do you have any protocols, advice or seen any publication where people have attempted this. Any input would be greatly appreciated!

Hi
I think i didnt get you what you mean is do chip using antibody and after getting chromatin you want to do wester to see other proteins in the complex ........is that right????
If so what antibodies you are going to use????//
Is it trial and error???? or what???

Hi!
thanks for your reply. I want to do CHIP and not just IP bevcause i want to pull down protein associated chromatin and hence relevant protein complex. We think its a multiple protein complex ( transcriptional activators or repressors depending on treatment to the cell line) associated with the chromatin. then we want to look at proteins associated by western. yes it is a first time experiment , so its s trial and error.

Hi,

I am also doing ChIP. I would be also interested in looking for other proteins. As far as I know after the wash in LiCL (Upstate protocol) you resuspend the beads in RIPA buffer and boil. Then you laod the supernatant to the SDS-gel. I haven`t tried it before -still problems in ChIP protocol itself What kind of ChIP protocol will you use?

Thanks

Hi zek
Thanks for replying! I am using a protocol from Current protocols which involves the usual cross linking , sonication and MNase treatment. i haven't started the CHIP protoccol , still babying my cells so that they grow well. ( i am having probelm with the cell line i need to do experiemnt in). I also looked at the upstate protcol and would be using it. i am attaching the protocol, i would be using mamalian cells
keep in touch so that we can brainstorm each other with problems.

Hi,

I am also doing ChIP. I would be also interested in looking for other proteins. As far as I know after the wash in LiCL (Upstate protocol) you resuspend the beads in RIPA buffer and boil. Then you laod the supernatant to the SDS-gel. I haven`t tried it before -still problems in ChIP protocol itself What kind of ChIP protocol will you use?

Thanks