Background in CHIP - (Apr/13/2005 )

Hi,

I have been doing CHIP with cultured cells for sometime now but have had a serious problem with background (in no Ab controls). It is strong in some samples and not there in others. Any suggestions on how I can reduce the background?

Thanks,
Shail

Hi Shail,

This post may help. It discusses the same problem as yours.

http://www.protocol-online.org/forums/inde...?showtopic=4646

thanks a lot ...
Shail

Hi
I have been trying to optimise ChIP for a non histone protein for the past year and I have been having a bit of trouble with the NA control too but found that using a primer set that amplifies a region downstream of my target region gives fairly consistent results for a negative control.....

Primer set downstream of my target gene also works for me as a negative control but it still is not the same as a No Antibody control....
I tried couple other things (increasing wash times etc.) but am still not very successful in reducing my background.

Does it help to use Protein G beads instead of Protein A beads (thats what I use as of now..).

Thanks,
S

Hi, I used to prehyb my Protein A with BSA and salmon sperm DNA. Have you tried this? It consistently gave me a reduced background level.

I also found varying degrees of noise from batch to batch and deoendent on how long the Protein A had been pre-swollen.

Spotty

Thank you. Prehyb with salmon sperm DNA seems to have helped...will know for sure after repeating this couple of times. Saw your note only last week (should have checked it sooner!).

Thank you,
S