EZ-ChIP™

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Just because an antibody works well in a Western blot does not always indicate it will perform well in chromatin immunoprecipitation. Unlike a Western blot that detects proteins that have been denatured, a ChIP antibody must recognize the target protein in its native state. Use ChIP-validated antibody. -Use 2-10 μg of your ChIP antibody depending on the abundance of your protein target.

Protocol tips
-Just because an antibody works well in a Western blot does not always indicate it will perform well in chromatin immunoprecipitation. Unlike a Western blot that detects proteins that have been denatured, a ChIP antibody must recognize the target protein in its native state. Use ChIP-validated antibody. -Use 2-10 μg of your ChIP antibody depending on the abundance of your protein target.

Publication protocol

Chromatin Immunoprecipitation (ChIP) was performed using the EZ‐ChIP Kit (Millpore, Temecula, CA, USA) as described in the manufacturer's protocol. Briefly, HEK293T cells were washed and fixed in culture medium containing 1% formaldehyde at room temperature for 10 min. The cells were collected and lysed to release the nucleus. Nuclei were then isolated by centrifugation before being subjected to sonication. The sonicated lysate was then immunoprecipitated with 2 μg of NRF‐1 antibodies (Abcam, New Territories, HK), or a negative control IgG at 4°C for 4 h. The pulled‐down chromatin was washed, reverse‐crosslinked and purified. Semi‐quantitative PCR was performed using 5% of precipitated chromatin. Primers targeting promoter sequences were shown in Table S2. PCR products were analyzed by quantitative real‐time PCR.

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Papers

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Manufacturer protocol

Download the product protocol from Merck Millipore for EZ-ChIP™ below.

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Videos

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