Anti h3k9ac antibody

Manufactured by Merck Group

The Anti-H3K9ac antibody is a laboratory reagent used for the detection and quantification of acetylated histone H3 at lysine 9 (H3K9ac) in various biological samples. This antibody is a valuable tool for researchers studying epigenetic regulation and chromatin dynamics.

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Lab products found in correlation

Protein g dynabead, by Thermo Fisher Scientific (1 mentions) Ez magna chip a g kit, by Merck Group (1 mentions) Anti h3k27me3 antibody, by Merck Group (1 mentions) Anti h3k4me3 antibody, by Merck Group (1 mentions) Anti h3k4me3, by Merck Group (1 mentions) Real time detection system, by Bio-Rad (1 mentions) Control igg, by Merck Group (1 mentions) Anti h3k27me3, by Merck Group (1 mentions) Ez magna chip kit, by Merck Group (1 mentions) Sybr premix ex taq 2 kit, by Takara Bio (1 mentions) Ideal chip seq kit for histones, by Diagenode (1 mentions) Spriselect beads, by Beckman Coulter (1 mentions) Hiseq platform, by Illumina (1 mentions)

Close spelling variants of this product

Rabbit anti-H3K9ac antibody H3K9ac antibody Anti-H3K9ac H3K9ac

6 protocols using anti h3k9ac antibody

ChIP-seq Analysis of Chromatin Modifications

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Synchronous parasites (12 hpi ± 3) were treated as above for the dCas9 ChIP with minor differences. 5 × 10⁸ parasites were cross‐linked and lysed in 4 ml ChIP lysis buffer. Chromatin was sonicated in 3.6 ml total ChIP SDS lysis buffer. For the ISWI‐HA ChIP, 8 μg DNA (chromatin) was diluted and incubated with 8 μg anti‐HA antibody (Abcam ab9110) conjugated to 200 μl Protein G Dynabeads (Invitrogen 10004D). For the H3K9ac ChIP, 2 μg DNA (chromatin) was diluted and incubated with 2 μg anti‐H3K9ac antibody (Millipore 07‐352) conjugated to 50 μl Protein G Dynabeads (Invitrogen 10004D).
Sequencing libraries were prepared, sequenced, and processed as with the dCas9 ChIP samples. Fold enrichment over input was calculated using deeptool's bamCompare (Ramírez et al, 2016) (option “–ratio ratio”) in windows of 1 nt (option “–bs 1”), and coverage plots were visualized in the Integrative Genomics Viewer (Robinson et al, 2011). Meta‐gene plots were visualized using deeptool's plotProfile over a region 1.5 kb upstream and downstream of the translation start and stop site, respectively, using the quality‐filtered bam files as input.

Bryant J.M., Baumgarten S., Dingli F., Loew D., Sinha A., Claës A., Preiser P.R., Dedon P.C, & Scherf A. (2020). Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite. Molecular Systems Biology, 16(8), e9569.

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ChIP Assay of Chromatin Modifications

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For ChIP assays, K562 cells were fixed with 1% (vol/vol) formaldehyde for 10 min at room temperature. ChIP assays were performed using the EZ-Magna ChIP A/G Kit (Millipore). Anti-Bmi1 antibody (Active Motif, AF27), Anti-Ring1b antibody (MBL International, D139-3), Anti-H3K9Ac antibody (Millipore, 07-942), Anti-H3K4me3 antibody (Millipore, 07-473), anti-H3K27me3 antibody (Millipore, 07-449), Anti-H2AK119ub1 antibody (Cell Signaling, D27C4) and normal mouse IgG were used for immunoprecipitation. ChIP DNA was then subjected to real-time PCR analysis using primers targeting different region of ribosomal protein gene promoters [23 (link)-24 (link)].

Gao R., Chen S., Kobayashi M., Yu H., Zhang Y., Wan Y., Young S.K., Soltis A., Yu M., Vemula S., Fraenkel E., Cantor A., Antipin Y., Xu Y., Yoder M.C., Wek R.C., Ellis S.R., Kapur R., Zhu X, & Liu Y. (2015). Bmi1 promotes erythroid development through regulating ribosome biogenesis. Stem cells (Dayton, Ohio), 33(3), 925-938.

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In vitro Acetylation Assay for Histone H3

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In vitro acetylation assay for rRv0428c was performed by using bacterially expressed recombinant histone H3 as substrate and acetyl-coA as a donor molecule and mammalian core histones as a positive control. The reaction was set up in acetyl-transferase assay buffer (50 mM Tris–Cl, pH 8, 10% glycerol, 10 mM butyric acid, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF) with 10 µM acetyl-coA, 10 µg histone H3, with rRv0428c. A time dependent kinetics was studied by incubating the reaction mixture for 1 h, 2 h and 3 h, in a 30 °C water bath followed by addition of 2X SDS-PAGE sample buffers and 10 min boiling for stopping the reaction. The prepared samples were then subjected to 18% SDS-PAGE gel electrophoresis and transferred onto PVDF membranes. The membrane was stained with 0.05% Fast green for validating equal loading of samples followed by western blotting with Anti H3K9ac antibody (1:5000, Millipore).

Sharma A., Kumar A., Rashid M., Amnekar R.V., Gupta S, & Kaur J. (2022). A Phagosomally Expressed Gene, rv0428c, of Mycobacterium tuberculosis Demonstrates Acetyl Transferase Activity and Plays a Protective Role Under Stress Conditions. The Protein Journal, 41(2), 260-273.

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Chromatin Immunoprecipitation and qPCR Protocol

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We performed qPCR to identify the genes using the same protocol as for the ChIP-seq experiments. Anti-H3K27me3, anti-H3K4me3, or anti-H3K9ac antibody, and control IgG (all from Millipore) were used for the ChIP assay. qPCR using SYBR green real-time PCR mixture (11202ES08, Yeasen, Shanghai, China) in a real-time detection system (Bio-Rad) was performed for the ChIP samples, with the primers listed in Supplementary Table 4.

Zhang Q., Kong W.L., Yuan J.J., Chen Q., Gong C.X., Liu L., Wang F.X., Huang J.C., Yang G.Q., Zhou K., Xu R., Xiong X.Y, & Yang Q.W. (2022). Redistribution of Histone Marks on Inflammatory Genes Associated With Intracerebral Hemorrhage-Induced Acute Brain Injury in Aging Rats. Frontiers in Neuroscience, 16, 639656.

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Chromatin Immunoprecipitation of H3K9ac and HDAC3

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Chromatin immunoprecipetition was carried out according to the protocol of the EZ-Magna ChIP kit (17-10086, Millipore). Briefly, the nuclear content of cortical neurons was extracted and the chromatin inside was sonicated into fragments. Fragmented chromatin was incubated and immunoprecipitated using anti-H3K9ac antibody (17-609, Millipore) and anti-HDAC3 antibody (17-10238, Millipore). After the de-crosslink, immunoprecipitated DNA and whole-cell extract DNA were then eluted and subjected to real-time PCR analysis using SYBR Premix Ex TaqII Kit (Takara, Shiga, Japan). All PCR primers are shown in Supplementary Table S2.

Yang X., Wu Q., Zhang L, & Feng L. (2016). Inhibition of Histone Deacetylase 3 (HDAC3) Mediates Ischemic Preconditioning and Protects Cortical Neurons against Ischemia in Rats. Frontiers in Molecular Neuroscience, 9, 131.

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Microglial H3K9ac ChIP-seq Analysis

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Anti H3K9ac ChIP-seq were performed using using the iDeal ChIP-seq kit for Histones (Diagenode, Cat.#C01010059) and NEBNext^® UltraTM II DNA Library Prep Kit for Illumina^® (NEB, Cat.#E7103S). Briefly, microglia were sorted from Itgb8^fl/fl;Emx1^Cre mouse brain cortex and were pooled from 3–4 different individuals with same genotype to achieve > 200 k cells per biological replicate. 3 mutant and 4 littermate control (Emx1-^Cre(-),Itgb8^fl/fl samples were generated in this way, from a total of 35 mice. The microglia were fixed with 1% formaldehyde at 20°C for 8 min then quenched with 0.125 M Glycine. The nuclei were isolated and sonicated using a Diagenode disruptor for 20 cycles (30 seconds “ON”, 30 seconds “OFF”, high power). The chromatin fragments were enriched using 2 ul of anti-H3K9ac antibody (Millipore, 07–352) per reaction. The ChIPed DNA was purified and processed for library preparation. The ChIP DNA libraries were size selected using SPRIselect beads (Beckman Coulter, Cat.#B23317) and quantified using Agilent 2100 Bioanalyzer and Qubit (Invitrogen, Cat.#Q33238). The pooled libraries were then sent to Azenta Life Sciences for paired-end sequencing (2×150 bp) on Illumina HiSeq platform.

McKinsey G.L., Santander N., Zhang X., Kleemann K., Tran L., Katewa A., Conant K., Barraza M., Waddell K., Lizama C., La Russa M., Koo H.J., Lee H., Mukherjee D., Paidassi H., Anton E.S., Atabai K., Sheppard D., Butovsky O, & Arnold T.D. (2023). Radial glia promote microglial development through integrin αVβ8 -TGFβ1 signaling. bioRxiv.

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