Histone H3K9me3 antibody (mAb)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-2 - 10 µg per ChIP. -Avoid over fixation. -purchase expensive, "hing end" sonicator to get reproducible shearing. -Avoid repeated freeze/thaw cycles. -Keep all reagents on ice when not in storage.

Protocol tips
-2 - 10 µg per ChIP. -Avoid over fixation. -purchase expensive, "hing end" sonicator to get reproducible shearing. -Avoid repeated freeze/thaw cycles. -Keep all reagents on ice when not in storage.

Publication protocol

ChIP assays were performed as described previously (16). Cells were cross-linked by using 0.5% formaldehyde, followed by treating with 125 mM glycine to quench the reaction. After washing with cold 1× PBS, cells were lysed for 10 min on ice in 1× CE buffer (10 mM HEPES-KOH [pH 7.9], 60 mM KCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM DTT and protease inhibitor tablet). The nuclei were pelleted by centrifugation at 700 × g for 10 min at 4°C and re-suspended in 1× SDS lysis buffer (1% SDS, 10 mM EDTA, 50 nM Tris-HCl [pH 8.1] and protease inhibitor mixture). Nuclear lysates were sonicated for 2 min to fragment genomic DNAs, and subsequently diluted to 10-fold with 1× ChIP buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl [pH 8.1], 150 nM NaCl and protease inhibitor mixture). The lysates were incubated for overnight at 4°C with specific antibodies or control mouse IgG or rabbit IgG. Protein A/G beads (Invitrogen) were pre-blocked with 0.5 mg/ml BSA and 0.125 mg/ml calf thymus DNA for 1 h at 4°C, and then added to the lysate-antibody mixture for another incubation at 4°C for 2 h. IPed samples were washed with the following buffers: low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl); high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl); LiCl buffer (0.25 M LiCl, 1% NP-40, 1% Na-deoxycholace, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]); and 1× TE buffer (10 mM Tris-HCl [pH 8.1], 0.1 mM EDTA)], and were eluted with 1× elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature. To reverse the cross-linking, the eluted samples were incubated at 65°C for overnight in the presence of 0.2 M NaCl. The eluted samples were then treated with proteinase K, and the DNA species were precipitated by using phenol–chloroform. The DNA pellets were re-suspended in water and quantified by qPCR. Input (1%) was used for qPCR analysis. The results were normalized to input values (percent input  =  2[(Ct, Input −  Ct, IP)  ×  100].

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Manufacturer protocol

Download the product protocol from Active Motif for Histone H3K9me3 antibody (mAb) below.

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