Please Help me, about Chromatin IP(ChIP)! - CHIP (Dec/29/2006 )

Hi all.

I want to use ChIP to study whether transcription factor(Sp1) binding to target sequence in Genome.
My ChIP protocol is according to the attachment file.
But unfortunately, I got significant background in IgG control.

Before ChIP assay, I have blocked proteinA beads in ssDNA/1% BSA solution.
And I tried to add washing time by using high salt buffer.
But it's not working.
I also tried to reduce PCR cycle to 22cycle, it doesn't working too.

I felt so sad because I tried all of improving method ing this forum.
Can anyother kind gentleman or lady give me some commet?

I'll deep appreciate your kindness.



Finally, happy new year!

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1) You should switch the order of your wash buffers and do low salt first. If you're worried that the salt from the high salt buffer will cause problems, you can just wash with TE, or something dilute, at the very end. In addition, many people do a lithium chloride wash as well.

2) You might try and see if diluting your chromatin before IP would help. Background drops off faster than the specific signal when you dilute.

3) This has nothing to do with your background problem but could make your life a bit easier. It seems that the old method for reversal of crosslinking (65C for several hours) and isolation of DNA (PCI and ethanol extraction) are not necessary. In our Fast ChIP method (Nucleic Acids Research 34(1): e2 and Nature Protocols 1(1) 179-185) a short digestion off the beads with prot K and boiling in Chelex-100 is sufficient for 100% the yield you would get with the old method.

Briefly, after washing the beads the last time (it's preferable if you do one last wash with TE) add 100ul of 10% chelex-100 suspension and 20ug of prot K. Incubate at 50C for 10-15min and then boil for 10-15min. Cool and take the supernatant. Add 100ul of H2O to the bead/chelex pellet, vortex, spin, and pool supernatant with the previous supernatant. Then run the PCR on the pooled supernatant. That's all.

Good luck and happy new year.

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