Histone H4K20me1 antibody (mAb)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-5 - 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
-5 - 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

Cells were collected at confluency or approximately 1 million cells/immunoprecipitation. Cells were washed with 1× PBS, cross-linked with 1% paraformaldehyde (ChemCruz) in PBS at room temperature, and quenched with 2.5 M glycine (Sigma Aldrich). Cells were then washed with ice cold PBS and lysed in 1 ml RIPA buffer (150 mM NaCl [Alfa Aesar], 1% v/v Nonidet P-40, 0.5% w/v deoxycholate [Amresco], 0.1% w/v SDS [Amresco], 50 mM Tris [Corning Cellgro] pH 8.0, 5 mM EDTA [Amresco]) plus inhibitors (leupeptin 1 μg/ml [Sigma Aldrich], aprotinin 1 μg/ml [Sigma Aldrich], pepstatin 1 μg/ml [Sigma Aldrich], benzamidine 1 mM [Sigma Aldrich], and PMSF 1 mM [Sigma Aldrich]). Lysates were sonicated using Diagenode Bioruptor with 25 cycles of 30-second pulses and 90-second intervals to shear DNA to ~500-bp fragments. Lysates were then cleared by centrifugation at 18,000 g for 15 minutes at 4°C. Protein A/G Sepharose beads (40 μl; MilliporeSigma) were added, and the lysate was precleared for 1 hour at 4°C. Protein A/G beads (20 μl) blocked with 1 mg/ml BSA was mixed with the precleared lysate, and immunoprecipitation was carried out by adding 5–10 μg of antibody (H4K20me1 [catalog 39727], H3K4me1 [catalog 39297]; Active Motif) rotating overnight at 4°C. Beads were washed 2× with RIPA, 4× with IP wash buffer (100 mM TrisHCL pH8.5, 500 mM LiCl, 1% v/v Nonidet-P-40, 1% w/v deoxycholic acid), and 2× with TE buffer. Immunocomplexes were eluted at 65°C with elution buffer (70 mM TrisHCl pH 8, 1 mM EDTA, 1.5% w/v SDS). The eluate was brought to a final concentration of 200 mM NaCl and reverse crosslinked at 65°C. The eluate was then treated with proteinase K (Invitrogen), and DNA was isolated by phenol/chloroform extraction (Invitrogen) and ethanol precipitation. ChIP-DNA was quantified using the Qubit dsDNA High Sensitivity Assay kit (Thermo Fisher Scientific).



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Manufacturer protocol

Download the product protocol from Active Motif for Histone H4K20me1 antibody (mAb) below.

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