Anti-Histone H3 (tri methyl K36) antibody - ChIP Grade (ab9050)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Add protease inhibitors to all lysis solutions before use.	-Use 4µg for 106 cells. -Keep cells on ice between the rounds of homogenisations. - Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line.

Upstream tips
-Add protease inhibitors to all lysis solutions before use.
Protocol tips
-Use 4µg for 106 cells. -Keep cells on ice between the rounds of homogenisations. - Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line.

Publication protocol

Sixty milligrams of each tissue was cut into 1-mm3 pieces in PBS with protease inhibitor. Tissue pieces were cross-linked for 10 min at room temperature with 1% formaldehyde and then quenched with 0.125 M of glycine for 5 min. Cross-linked tissues were triturated by trituration equipment for 30 s and then centrifuged at 12000 rpm, 4 °C for 5 min. Supernatant with massive oil was discarded and the precipitates were lysed with 1 mL lysis buffer (50 mM Tris-HCl pH 8.0, 0.1% SDS, 5 mM EDTA) and incubated for 5 min with gentle rotation. After centrifugation at 12000 rpm, 4 °C for 2 min, lysates were washed once by digestion buffer (50 mM Tris-HCl pH 8.0, 1 mM CaCl2, 0.2% Triton X-100). Washed lysates were incubated in 630-μL digestion buffer with 1-μL MNase (NEB, M0247S) at 37 °C for 20 min and then quenched with 8 μL 0.5 M EDTA. Whole lysates were sonicated and the supernatants were taken out after centrifugation. Thirty microliters of supernatants were taken for checking the efficiency of MNase digestion. Immunoprecipitation was further performed with 150-μL sheared chromatin, 2-μg antibody, 50-μL Protein G sepharose beads and 800-μL dilution buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS) overnight at 4 °C. Next day, immuno-complexes were washed once with Wash buffer I (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), once with Wash buffer II (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), once with Wash buffer III (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% Na-deoxycholate, 1% NP-40), and twice with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The immune complexes were eluted twice with 100-μL elution buffer (1% SDS, 0 .1M NaHCO3, 20 mg/mL Proteinase K) at room temperature. The elution was incubated at 65 °C for 6 h and then purified with DNA purification kit (TIANGEN DP214-03).

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Manufacturer protocol

Download the product protocol from Abcam for Anti-Histone H3 (tri methyl K36) antibody - ChIP Grade (ab9050) below.

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