Anti-monomethyl-Histone H3 (Lys4) Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Immunoprecipitations were carried out in 1 ml of IP buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 % Triton-X 100). Three µg antibody was used per 20 µg purified chromatin. Input chromatin was obtained after preclearing by de-crosslinking and purified using the Qiaquick column (Qiagen) according to manufacturer’s instructions. Immunoprecipitations were carried out with inverting at 4 °C for 14–16 h

Protocol tips

Immunoprecipitations were carried out in 1 ml of IP buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 % Triton-X 100). Three µg antibody was used per 20 µg purified chromatin. Input chromatin was obtained after preclearing by de-crosslinking and purified using the Qiaquick column (Qiagen) according to manufacturer’s instructions. Immunoprecipitations were carried out with inverting at 4 °C for 14–16 h

Publication protocol

Infected RBCs were crosslinked with 1 % formaldehyde (Catalog number—28908, THERMO Scientific) for 10 min, lysed and sonicated in sonication buffer (10 mM Tris–HCl pH 7.5, 200 mM NaCl, 1 % SDS, 4 % NP-40, 1 mM PMSF) to obtain an average chromatin size of 200–400 bp. Chromatin was pre-cleared using 50 µl of a 50 % protein A Sepharose (GE healthcare) slurry for 1 h at 4 °C with gentle inverting. Immunoprecipitations were carried out in 1 ml of IP buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 % Triton-X 100). Three µg antibody was used per 20 µg purified chromatin. Input chromatin was obtained after preclearing by de-crosslinking and purified using the Qiaquick column (Qiagen) according to manufacturer’s instructions. Immunoprecipitations were carried out with inverting at 4 °C for 14–16 h. The samples were then incubated with 50 µL of a 50 % Protein A Sepharose slurry for 3 h at 4 °C with gentle inverting. IP samples were reverse-crosslinked and the DNA was purified using a Qiaquick column (Qiagen). Target sites obtained from ChIP-seq analysis were further validated by quantitative PCR using Power SYBR Green Master Mix (Applied Biosystems). For sequential ChIP, at least four ChIP assays (4 × 20 µg purified chromatin) were used for the first IP (H3K9ac). Following standard washing, elution was performed with 10 mM DTT (30 min, 37 °C). The eluates from four ChIPs were combined, diluted at least 30 times with ChIP dilution buffer and secondary antibody (H3K4me3) was incubated overnight. The subsequent steps were performed as for regular ChIP.

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 Anti-bodies H3K4me1 using Anti-monomethyl-Histone H3 (Lys4) Antibody from Merck Millipore.

Manufacturer protocol

Download the product protocol from Merck Millipore for Anti-monomethyl-Histone H3 (Lys4) Antibody below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing ChIP Anti-bodies H3K4me1 using Anti-monomethyl-Histone H3 (Lys4) Antibody 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.