Chromatin Immunoprecipitation (ChIP) Assay Kit

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

ChIP assays were carried out with a EZ-ChIP kit (Millipore, Billerica, MA, USA) per
manufacturer’s protocol. Briefly, three biological replicates of cardiac fibroblast from
ATF3KO mice transfected with Ad-con or Ad-ATF3 for 48 hours were treated with Ang
II for 6 hours. Cells were cross-linked with 1% paraformaldehyde, before lysis with
cold PBS/protease inhibitor. Chromatin was sheared by 10 seconds of sonication
(Covaris 220) interposed with 30 second pauses, repeated 10 times.
Immunoprecipitation was performed overnight at 4°C, via 4 μg of specific antibody or
immunoglobulin G. Each IP sample was added to protein G agarose beads for 3 hours
incubation at 4 °C. After pull-down, beads were washed 5 times. The
immunocomplexes were extracted and processed by reverse cross-linking, proteinase
K digestion, and DNA precipitation. The following antibodies were used for Chip:
rabbit anti-ATF3 antibody (C-19, Santa Cruz), rabbit anti-acetyl-Histone H4 antibody
(Millipore), and Ig G (Sigma). The following primers were used for Map2K3 ChipPCR: forward 5'-CAGGACCACGCCAGCACAATC-3' and reverse 5'-
CGGACTGTCACGCACAAACCC-3'.


Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing ChIP Mouse - Cardiac fibroblasts using Chromatin Immunoprecipitation (ChIP) Assay Kit from Merck Millipore.

Manufacturer protocol

Download the product protocol from Merck Millipore for Chromatin Immunoprecipitation (ChIP) Assay Kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing ChIP Mouse - Cardiac fibroblasts using Chromatin Immunoprecipitation (ChIP) Assay Kit from Merck Millipore. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.