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

A chromatin immunoprecipitation (CHIP) kit (EZ-ChIP™; #17–371; EMD Millipore) was used for a quantitative chromatin immunoprecipitation (qChIP) assay, based on the manufacturer’s manual. In brief, we fixed NRK52E transfected with pGC–LV–PAX2 using 1% paraformaldehyde, and then sheared chromatin derived from isolated nuclei. Each incubation mixture contained 100 µL of sheared chromatin, 60 µL protein G agarose beads, and 10 µg PAX2 antibodies (Abnova, Taipei, Taiwan). We used the following primer pairs, 5′-CATTGGGTACGGATGCGTCAC-3′ and 5′-GCCTC TTCCTTCCTCCCTTCC-3′, to amplify a 119-bp product of the human ADAM10 promoter containing the putative PAX2 binding site (for sequence, referred to NM_001110.3).

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Manufacturer protocol

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

Download manufacturer protocol

Videos

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