Chromatin Immunoprecipitation (ChIP Assay) - Protocol

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Divaker Choubey

see the link below for a highly optimized protocol:

http://farnham.genomecenter.ucdavis.edu/pdf/FarnhamLabChIP%20Protocol.pdf

Luis André Mariuba

Sorry, I never done this kind of immunoprecipitation. Try the Antibody lab manual (Harlow and Lane), maybe classical immunoprecip. could help you in some way.

wen-rong Lie

I have not done this assay myself but I know EMD Millipore has an epigenetics portfolio with many ChIP reagents and assay kits. You can visit the www.millipore.com website, type in "ChIP", then open the "Technical Library" tag, you can find ChIP protocols there (e.g. http://www.millipore.com/userguides/tech1/mcproto407). You can also try to call Millipore technical service if you have specific questions on ChIP.

Ashok Sharma

Hi,

I did lot of ChiP. Please purchase your choice of kit from millipore (eg.EZ-ChIP™) and follow kit protocol. I tell some point where you take extra care:

1. Fixed the live cell with formaldehyde at least 15'

2.After fixing sonicate the cells with sonicator 15' pulse at least 30 times on dry ice (take care sample are not heatup) after sonication check the DNA on agarose gel that all DNA sonicated well or not (means DNA ladder come between 100bp to 500bp) if not please again do sonication. This step is very-very important for chip and need to standarization according to cells used.

3. Digested the protein with proteinase-K very well at least 2hr at 60oC on rotating strand.

During all protocol uses 4oC temp and usesd proteinase inhibitor at all required steps.

good luck!!:)

ashok

Aprajita Tiwari

I have not done it but i can suggest check out:

reference:http://faculty.virginia.edu/mirmira/resources_files/Protocols/ChIP%20Assay%20Protocol.htm

Jacques Serratrice

Sorry! I have never done this assay.

You Could ask Pierre Cauchy (Inserm, U928, TAGC, Marseille, France).

Anna Yarilina

Hi Mustafa,

Specific conditions for the ChIP such as fixing time, sonication protocol or washing conditions are different for different cell types, your protein of interest (transcription factors vs histones ) and other factors. I would recommend to look at the methods in papers describing system close enough to yours. You can learn about basic principle of ChIP using technical resources from companies such as Millipore, Abcam, Active Motif or on the internet. Good luck!