Histone modification-ChIP analysis on Fibroblasts - (Feb/06/2008 )

Hi everybody,

I'm studying Histone modifications by using the ChIP analysis on Fibroblasts.
I'm using the Upstate kit, but my whole analysis looks crap.
My IP- (no ab IP), gave specific product in the Real-Time PCR, with ct-value of 25.
I'm using anti-acetyl H4 for an actif gene and anti-trimethyl H3 Lys 9 for an inactif gene.
But I can not see this in my analysis whitch celline contains the active gene and witch the inactive.
De ct-values of the IP samples are around 25.

Does have anybody experience with ChIP analysis on Fibroblasts?
And have somebody any advice to doe?

Thanks!!

Carola

-Carola82-

QUOTE (Carola82 @ Feb 6 2008, 01:23 AM)

One of the things we've found to be most helpful in our ChIP assays using mammalian cell lines is to titrate the amount of chromatin used in order to reduce the background (the signal in your no Ab control). If you are saturating your antibody with chromatin, then reducing the amount of chromatin you use will decrease the no Ab signal while not affecting the signal of your specific IP. Considering the low Ct value for your no Ab control I would guess that this is your problem.

-KPDE-

Thanks, I will try to decrease the amount of chromatin.

-Carola82-