For DNA dot blot analysis, total DNA was extracted, quantified. DNA (80 ng) was loaded on nitrocellulose membrane, air dried, and exposed in UV for 20 min. After blocking with by 5% BSA/TTBS at room temperature for 2 hours, the membrane was incubated in anti-5hmC (Active Motif) and anti-5mC (Abcam) antibodies at 4°C overnight. For protein blot, histones were acid-extracted from cell samples. 2µg of histone samples were electrophoresized with 15% SDS-PAGE gels, and the blotted membranes were incubated with anti-H3K4m3, H3K9m3, H3K27m3, H3K36m3 and H3 antibodies (all from Millipore). Positive bands were detected and captured by ChemiDocR (Bio-rad, Hercules, Ca), and intensities of the bands were quantified using Image J software (http://imagej.nih.gov/ij ). Quantification was performed using average values from three independent experiments.
H3k27m3
Manufactured by Merck Group
H3K27m3 is a laboratory reagent used for the detection and quantification of trimethylation of lysine 27 on histone H3 in biological samples. It is a tool for epigenetic research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Antih3k4m3, by Merck Group (1 mentions)
Anti 5mc, by Abcam (1 mentions)
Anti 5hmc, by Active Motif (1 mentions)
Anti flag, by Cell Signaling Technology (1 mentions)
Anti ha, by Merck Group (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Suz12, by Abcam (1 mentions)
Ezh2 d2c9, by Cell Signaling Technology (1 mentions)
Actin, by Merck Group (1 mentions)
3 protocols using h3k27m3
1
Quantifying DNA Modifications and Histone Marks
2
Protein Interactions in Circadian Regulation
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HEK293T cells were seeded in 10-cm Petri dishes. After reaching 90% confluency, cells were transfected with 6 ug each of bmal1b-HA or bmal1b-His, clock1a-Flag and ezh2-His or ezh2-HA, and 2 ug of cry1aa-His. Forty eight hours after transfection, cells were lysed in RIPA buffer with protease inhibitor (Sigma) and subjected to co-immunoprecipitation (Co-IP) according to manufacturer's instructions (Sigma). Briefly, cell lysates were released with protein G–Sepharose beads conjugated with anti-HA antibodies. After washing five times, the precipitates were re-suspended in SDS–PAGE sample buffer, boiled for 5 min and run on 12% SDS–PAGE gel, and then western blotting (WB) was conducted with mouse monoclonal anti-His (Cell Signaling Technology), anti-HA (Sigma) or anti-Flag (Cell Signaling Technology) antibody or rabbit polyclonal anti-Tubulin antibody. To detect the protein levels in zebrafish embryos, 48-hpf wild-type and ezh2–/– mutant embryos were collected and lysated with RIPA buffer, respectively. Equal amount of wild-type and ezh2 embryo extracts were loaded on SDS-PAGE, and then WB was performed using antibodies against EZH2 (Millipore), Eed (Millipore), H3K27m3 (Millipore), H3K27m2 (Millipore), H3K27m1 (Millipore), H3 (Cell Signaling Technology).
3
Chromatin Isolation and Western Blot Analysis
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Whole-cell lysates and nuclear lysates were prepared using RIPA buffer and RSB buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, and inhibitors), respectively, as previously described19 (link). Protein lysates were resolved by SDS–PAGE and transferred onto nitrocellulose membranes. Antibodies used were the following: EZH2 (D2C9; Cell signaling), SUZ12 (ab12073; Abcam), H3K27m3 (07–449; Millipore), HP1γ (clone 42S2; Millipore), and ACTIN (A5441; Sigma-Aldrich). Following secondary antibody incubations, signals were visualized by enhanced chemiluminescence. Full gels can be found in Supplementary Fig. 9 .
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