ChIP was performed as previously described58 (link). For each sample, 30 μl of Protein A Dynabeads (Invitrogen) and 2 μg anti–histone H3-dimethyl K9 (Abcam 1220) antibody were used. Validation information for anti–histone-H3-dimethyl K9 is provided on the manufacturer’s website.
Anti histone h3 dimethyl k9
Manufactured by Abcam
Anti–histone H3-dimethyl K9 is a lab reagent that specifically binds to histone H3 proteins that have been dimethylated at the lysine 9 (K9) residue. This reagent can be used to detect and study this histone modification, which is associated with gene silencing and the formation of heterochromatin.
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Lab products found in correlation
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Thermo Fisher Scientific (1 mentions)
Ez magna chip a chromatin immunoprecipitation kit, by Merck Group (1 mentions)
Mj opticon 2 thermo cycler, by Bio-Rad (1 mentions)
Anti acetyl histone h3, by Merck Group (1 mentions)
Chipab trimethyl histone h3 lys4, by Merck Group (1 mentions)
Platinum sybr green qpcr supermix udg, by Thermo Fisher Scientific (1 mentions)
Anti histone h3, by Abcam (1 mentions)
3 protocols using anti histone h3 dimethyl k9
1
ChIP Assay for H3K9 Dimethylation
2
Chromatin Immunoprecipitation in CD4+ T Cells
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ChIP was carried out using the EZ-Magna ChIP A Chromatin Immunoprecipitation Kit (Millipore). A total of 20 × 106 CD4+ T cells were fixed in 1% formaldehyde (Fisher Scientific). Glycine buffer was added to quench any remaining formaldehyde. Cells were then lysed and sonicated using a Diagenode bath sonicator at high setting with 30-s pulses over a 5-min period. Immunoprecipitation was carried out using the following antibodies: anti-acetyl-histone H3 (Millipore), ChIPAb+ Trimethyl-Histone H3(Lys4) (Millipore), ChIPAb+ Trimethyl-Histone H3(Lys27) (Millipore), anti-Histone H3 (di methyl K9) (Abcam), and rabbit IgG (Millipore). Real-time PCR analysis was carried out using the MJ Opticon 2 thermo cycler (MJ Research) and Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). Cycling conditions were set up according to the manufacturer's instructions; annealing temperature for all primers was 55°C with 40 cycles of amplification. For ChIP primers and statistical analysis, see Supporting Information Method 2.
3
Histone Modification Antibody Analysis
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We used the following specific antibodies in this study: polyclonal rabbit anti-human anti-histone H3 tri methyl K79 (Abcam), anti-histone H3 di methyl K27 (Abcam), anti-histone H4 mono methyl K20 (Abcam), anti-histone H3 di methyl K9 (Abcam), and anti-histone H3 (Abcam).
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