Anti-trimethyl-Histone H3 (Lys4) Antibody, clone MC315, rabbit monoclonal

ChIP Anti-bodies H3K4me3

Experiment
ChIP Anti-bodies H3K4me3
Product
Anti-trimethyl-Histone H3 (Lys4) Antibody, clone MC315, rabbit monoclonal from Merck Millipore
Manufacturer
Merck Millipore

Protocol tips

Protocol tips
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).

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).

Full paper   Login or join for free to view the full paper.

Reviews

Anti-trimethyl-Histone H3 (Lys4) Antibody, clone MC315, rabbit monoclonal from Merck Millipore has not yet been reviewed for this experiment

We'd love it if you would be the first to write a review!

Discussion

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing ChIP Anti-bodies H3K4me3 using Anti-trimethyl-Histone H3 (Lys4) Antibody, clone MC315, rabbit monoclonal from Merck Millipore.

View full paper   Login or join for free to view the full paper.

Manufacturer protocol

Download the product protocol from Merck Millipore for Anti-trimethyl-Histone H3 (Lys4) Antibody, clone MC315, rabbit monoclonal below.

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing ChIP Anti-bodies H3K4me3 using Anti-trimethyl-Histone H3 (Lys4) Antibody, clone MC315, rabbit monoclonal 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.

We haven't found any additional videos for this experiment / product combination yet.

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms