Anti h3k9ac antibody

Manufactured by Cell Signaling Technology

Sourced in United States

The Anti-H3K9Ac antibody is a laboratory reagent used to detect acetylation of lysine 9 on histone H3 protein. It can be used in various techniques, such as Western blotting, immunoprecipitation, and chromatin immunoprecipitation, to study epigenetic modifications.

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Lab products found in correlation

Bicinchoninic acid colorimetric assay system, by Merck Group (2 mentions) His tagged histone h3, by BPS Biosciences (2 mentions) Hoechst 33342, by Cell Signaling Technology (1 mentions) Alexa 488 secondary antibody, by Cell Signaling Technology (1 mentions) Anti h3k27ac antibody, by Cell Signaling Technology (1 mentions) Chip assay kit, by Merck Group (1 mentions) Anti p65 antibody, by Cell Signaling Technology (1 mentions) Ez chip chromatin immunoprecipitation kit, by Merck Group (1 mentions)

5 protocols using anti h3k9ac antibody

In Vitro Histone Acetylation Assay for Chromatin

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In vitro histone acetylation assays were performed on 25 μg of mitotic chromatin (isolated as described above and quantified using the bicinchoninic acid colorimetric assay system [Sigma-Aldrich]) in 50 mM Tris–HCL, 10% glycerol, 1 mM DTT, 1 mM PMSF, 50 nM TSA, 0.1 mM EDTA supplemented with 100 μM Acetyl-CoA, and 10 μg His-tagged histone H3 (#52023; BPS Bioscience). The reaction was incubated at 37°C for 1 h. HAT activity was quantified by: (i) a colorimetric assay quantifying the release of Co-A using DTNB (2-nitrobenzoic acid) (Sigma-Aldrich) by measuring the absorbance at 412 nm; (ii) isolating the His-tagged H3 by Ni-NTA column and measuring H3K9ac by immunoblots using anti-H3K9Ac antibody (Cell Signalling #C5B11; 1:1,000). All acetylation assays were performed in duplicate.

Gandhi S., Mitterhoff R., Rapoport R., Farago M., Greenberg A., Hodge L., Eden S., Benner C., Goren A, & Simon I. (2022). Mitotic H3K9ac is controlled by phase-specific activity of HDAC2, HDAC3, and SIRT1. Life Science Alliance, 5(10), e202201433.

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Examining Histone H3K9ac Levels

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MEC cells were seeded in 6-well plates (5×10⁴ cells) with DMEM/High glucose supplemented as previously described and treated with Cephaeline (IC₅₀) in triplicates for 24 and 48h. After, cells were fixed with formaldehyde 4% for 15 min at room temperature. Blockage and cellular permeabilization were performed with 3% (w/v) bovine serum albumin (BSA) and 0.5% (v/v) Triton X-100 in PBS 1X for 1h. Anti- H3K9ac antibody (Cell Signaling Technology, Danvers, MA, USA) was diluted in (0.5% (v/v) Triton X-100 in PBS 1X and 1% (w/v) BSA) and incubated overnight. Subsequently, cells were washed and incubated with Alexa 488 secondary antibody (Cell Signaling Technology, Danvers, MA, USA) following with DNA staining using Hoechst 33342 (Cell Signaling Technology, Danvers, MA, USA). Ten fields of each slide were photographed and quantified. Images were taken using Nikon Eclipse Ti-S microscope and evaluated using Image J software (National Institute of Health, Bethesda, Maryland, USA).

da Silva L.C., Borgato G.B., Wagner V.P., Martins M.D., Rocha G.Z., Lopes M.A., Santos-Silva A.R., de Castro Júnior G., Kowalski L.P., Nor J.E., Squarize C.H., Castilho R.M, & Vargas P.A. (2021). Cephaeline is an inductor of histone H3 acetylation and inhibitor of mucoepidermoid carcinoma cancer stem cells. Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology, 51(6), 553-562.

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ChIP Assay for Immune Cell Epigenetics

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T cells were harvested and cross-linked according to the ChIP Assay Kit (Millipore) protocol. ChIP assay was performed with anti-Foxo1 antibody, anti-H3K9Ac antibody, anti-H3K27Ac antibody, anti-H3K4me3 antibody or control normal rabbit IgG antibody (Cell Signaling Technology), anti-RORγt (AFKJS-9) antibody (Thermo Fisher Scientific), and normal rat IgG antibody (Santa Cruz Biotechnology). The primers for qPCR were listed as follows: lncRNA-GM, GAA TTT TGT GGC AGC TCA GC (forward) and ACA CAC AAT AGC CTT GGC TG (reverse); Il23r, CAC CAT TCG CCC TCA AGA AC (forward) and CGT CTC TGG AGG TCA TGG TT (reverse); Il17, AGC TCC CAA GAA GTC ATG CT (forward) TAC GTC AAG AGT GGG TTG GG (reverse); Foxp3, GTT CCT GCT GTC AGG GTA GC (forward) and GTT CTT GTC AGA GGC AGG CT (reverse).

Chen Y., Liu J., Zhang X., Zhu H., Wang Y., Li Z., Liu Y., Liu S., Liu S., Li N., Chen K, & Cao X. (2022). lncRNA-GM targets Foxo1 to promote T cell–mediated autoimmunity. Science Advances, 8(31), eabn9181.

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ChIP-qPCR Assay for Investigating Transcription Factor Binding and Histone Modifications

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An EZ‐CHIP chromatin immunoprecipitation kit (Millipore) was utilised for chromatin immunoprecipitation (ChIP) assays. At the beginning, cells were cross‐linked with 1% formaldehyde. After quenching, the paraformaldehyde reaction with glycine, cells were lysed with lysis buffer containing Protease Inhibitor Cocktail II and sonicated on ice for chromatin immunoprecipitation assays. Ten percent of the supernatant was saved as the input fraction. The rest was used for immunoprecipitation with 5 µg of anti‐p65 antibody (Cell Signalling Technology, #8242), anti‐H3K9Ac antibody (Cell Signalling Technology, #9649) or anti‐rabbit IgG. DNA was purified and quantified by RT‐qPCR. IgG was included as nonspecific control. The results were calculated using % input = 2^(‐ΔCt [Ct [p65] – Ct [input]]) method. The primers for ChIP‐qPCR are shown in Table S2.

Jia X., Wang G., Wu L., Pan H., Ling L., Zhang J., Wen Q., Cui J., He Z., Qi B., Zhang S., Luo L, & Zheng G. (2023). XBP1‐elicited environment by chemotherapy potentiates repopulation of tongue cancer cells by enhancing miR‐22/lncRNA/KAT6B‐dependent NF‐κB signalling. Clinical and Translational Medicine, 13(1), e1166.

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Histone Acetylation Quantification Assay

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In vitro histone acetylation assays were performed on 25 μg of mitotic chromatin (isolated as described above and quantified using the bicinchoninic acid colorimetric assay system (Sigma)) in 50 mM Tris-HCL, 10% glycerol, 1mM DTT, 1mM PMSF, 50nM TSA, 0.1 mM EDTA supplemented with 100μM Acetyl-CoA and 10 μg Histagged Histone H3 (BPS Bioscience #52023). The reaction was incubated at 37°c for 1 h. HAT activity was quantified by: i) a colorimetric assay quantifying the release of Co-A using DTNB (2-nitrobenzoic acid) (Sigma) by measuring the absorbance at 412nm; ii) isolating the His-tagged H3 by Ni-NTA column and measuring H3K9ac by immunoblots using anti-H3K9Ac antibody (Cell Signalling # C5B11; 1:1000). All acetylation assays were performed in duplicate.

Gandhi S., Mitterhoff R., Rapoport R., Farago M., Greenberg A., Hodge L., Eden S., Benner C., Goran A., & Simon I. (2021). Mitotic H3K9ac is controlled by phase-specific activity of HDAC2, HDAC3 and SIRT1.

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