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Chromation Immunoprecipitation


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#1 lachlanmcdonald

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Posted 15 October 2003 - 11:02 PM

Hi,

I intend to start doing some ChIP assays from culture cells initially then from mouse tissue.

Given the difficult nature of ChIP assays I'd greatly appreciate any advice/ protocols on the system, including caveats.

Thanks very much,

Lachlan

#2 pcrman

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Posted 16 October 2003 - 09:30 AM

Hi Lachlan,

I think the critical part is the DNA sonication. You have to determine the proper settings for shearing DNA before the actual experiment. I used the ChIP kit and antibody from Upstate. You can find the protocol on their site (http://www.upstatebiotech.com) (no affilation with them). By using that kit, it's pretty easy to carry out the exp.

Good luck.

pcrman

#3 logie

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Posted 20 October 2003 - 10:48 AM

Active Motif have also just released a new ChIP kit (that includes an antibody known to work to TFIIB) that they say is better than other kits, but I've not tried it - I use home-made solutions etc.

I recommend testing your antibody first on small scale if you're not sure of its suitability. You might find with some antibodies that you need lots of cells or lots of antibody to recover a decent amount of chromatin.

Also, you may have to fiddle with PCR conditions to amplify your product. Some require lots of PCR cycles - probably reflecting poor recovery by the antibody. I recommend fiddling around with some genomic DNA (The DNA inputs for example) to optimise the PCR thoroughly using a temp gradient coupled with a hot PCR - that will enable you to amplify as best as possible anything you get back form the IP.

good luck !

logie




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