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  • Characterization of Epigenetic Histone Activation/Repression Marks in Sequences of Genes by Chromatin Immunoprecipitation-Quantitative Polymerase Chain Reaction (ChIP-qPCR).

Characterization of Epigenetic Histone Activation/Repression Marks in Sequences of Genes by Chromatin Immunoprecipitation-Quantitative Polymerase Chain Reaction (ChIP-qPCR).

Methods in molecular biology (Clifton, N.J.) (2019-05-10)
Shama Bhatia, Jason Matthews, Peter G Wells
ABSTRACT

Chromatin immunoprecipitation (ChIP) is widely used to measure protein-DNA interactions. This protocol outlines a ChIP method used to identify the association of a protein or protein modification (such as a specific histone modification-methylation, acetylation, etc.) of interest with a specific DNA sequence in a target gene in fetal mouse brains on gestational day (GD) 17. Briefly, DNA and proteins are cross-linked (via formaldehyde), and chromatin is sonicated into fragments between 200 and 1000 base pair (bp) long, with an average length of 500 bp. The DNA-protein complexes are captured using antibodies directed toward the protein or protein modification of interest. These immunoprecipitated complexes are retrieved using agarose beads. The DNA-protein cross-links are reversed (via heat and via presence of high salt concentrations), and the ChIP DNA is purified and measured via a quantitative polymerase chain (qPCR) reaction. The results show the association of histone modifications at unknown sites of specific genes of interest, indicating which epigenetic modifications of specific genes may be responsible for the outcome of interest.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-acetyl-Histone H3 (Lys9) Antibody, Upstate®, from rabbit
Roche
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail, Protease Inhibitor Cocktail Tablets provided in a glass vial, Tablets provided in a glass vial
Sigma-Aldrich
Anti-trimethyl-Histone H3 (Lys27) Antibody, Upstate®, from rabbit
Sigma-Aldrich
Rabbit IgG, Plasma
Sigma-Aldrich
Anti-Histone H3 Antibody, CT, pan, serum, Upstate®
Sigma-Aldrich
Protein A Agarose/Salmon Sperm DNA, 2.5 mL, for use in chromatin immunoprecipitations (ChIP assays)