Help- Background in ChIP - Can't get rid of background (Jul/01/2005 )

Hi All,

My problem is that i cannot seem to get rid of the non-specific background. I get bands of the right size being amplified along with everything else. I have tried longer pre-clearing times and extra washes, but i can't see much change.

I have tried the Upstate kit, and am now using a modified ChIP protocol gathered from several sources. I've recently started pre-binding pre-immune serum to my protein-G beads overnight before pre-clearing. Has anyone done this before?

Any advice would be greatly appreciated!

babynekko

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Hi babynekko,

I am not too sure what you mean when you say you get the band of interest but with everything else.....what is everything else? are you talking PCR? or westerns of your IP fractions?


Cheers

nick

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Hi nick,

I do PCR amplification. What i meant was that i get a band of the right amplicon size in my negative control which is at times alot darker than the antibody pull-down.

babynekko

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hmmmm,

when you meannegative control, is this your no-ab fraction control? or no-template control?

Nick

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this is the pre-immune/normal serum control, sort of like a no-ab control.

babynekko

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with your antibody, is it a ChIP grade one? if not have you tried it on a western to check if it binds to the protein of interest?

We check all antibodies (including ChIP grade ones) on a western blot and by immunofluorescence before embarking on a ChIP experiment. This is the make sure the antibody is doing what it is supposed to be doing.

Nick

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