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H3k27ac antibody

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The H3K27ac antibody is a lab reagent used to detect and analyze the acetylation of lysine 27 on histone H3 protein. It is a tool for studying epigenetic gene regulation and chromatin dynamics.

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Lab products found in correlation

Accel ngs 2s plus dna library kit, by Integrated DNA Technologies (5 mentions) H3k4me3 antibody, by Abcam (4 mentions) Bioruptor, by Diagenode (3 mentions) Hiseq 3000, by Illumina (3 mentions) Ovation ultralow library system v2, by Tecan (3 mentions) E220 sonicator, by Covaris (3 mentions) Yy1 antibody, by Santa Cruz Biotechnology (2 mentions) Bioruptor pico sonication device, by Diagenode (2 mentions) Dynabeads protein a, by Thermo Fisher Scientific (2 mentions) H3k4me3 antibody, by Merck Group (2 mentions) Dynabeads protein g beads, by Thermo Fisher Scientific (2 mentions) S220 focused ultrasonicator, by Covaris (2 mentions) Truchip high cell chromatin shearing kit with sds shearing buffer, by Covaris (2 mentions) Sx 8g ip star automated system, by Diagenode (2 mentions) Dynabeads protein g, by Thermo Fisher Scientific (2 mentions) H3k27me3 antibody, by Cell Signaling Technology (2 mentions) Qiaquick pcr purification kit, by Qiagen (2 mentions) Minelute pcr purification kit, by Qiagen (2 mentions) Thruplex dna seq kit, by Takara Bio (2 mentions) Hiseq 4000, by Illumina (2 mentions)

Close spelling variants of this product

H3K27ac (231 citations) Anti-H3K27ac antibody (28 citations) Anti-H3K27Ac (ab4729) antibody (3 citations) Histone H3K27Ac antibody (2 citations) H3K27Ac antibody Ab4729 (2 citations)

65 protocols using h3k27ac antibody

1

ChIP-seq Profiling of H3K27ac and YY1

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The ChIP protocol was conducted as described by Schmidt et al.58 (link) with few modifications. In summary, following fixation, the tumour tissue undergoes chromatin extraction and sonication using the Bioruptor Pico sonication device (Diagenode; B01060001) using 20 cycles (30s on and 30s off) at maximum intensity. Purified chromatin was then separated for 1. Immunoprecipitation using 4ug of H3k27ac antibodies (Abcam; ab4729) per ChIP experiment or using 4ug of YY1 antibodies (Santa Cruz; sc-281 X). ChIP-seq experiment for YY1 were performed in biological duplicates. Cells were stimulated with estrogen for 45 minutes, upon which maximum ERα-binding to chromatin occurs. Biological replicates showed very high correlation (R2=0.98), thus only consensus loci were kept for further analyses. 2. Non-immunoprecipitated chromatin, used as Input control and 3. Assessment of sonication efficiencies using a 1% agarose gel. Before construction of ChIP-seq libraries (NEB Ultra II kit, see supplementary methods), enrichment of the immunoprecipitated sample was ascertained using positive and negative controls for ChIP-qPCR. Library preparation was performed using 10 – 50 ng of immunoprecipitated and Input samples. Before sequencing, libraries were again re-tested to confirm enrichment using positive and negative controls.
Patten D.K., Corleone G., Győrffy B., Perone Y., Slaven N., Barozzi I., Erdős E., Saiakhova A., Goddard K., Vingiani A., Shousha S., Pongor L.S., Hadjiminas D.J., Schiavon G., Barry P., Palmieri C., Coombes R.C., Scacheri P., Pruneri G, & Magnani L. (2018). Enhancer mapping uncovers phenotypic heterogeneity and evolution in patients with luminal breast cancer. Nature medicine, 24(9), 1469-1480.
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2

ChIP-seq Profiling of H3K27ac and YY1

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The ChIP protocol was conducted as described by Schmidt et al.58 (link) with few modifications. In summary, following fixation, the tumour tissue undergoes chromatin extraction and sonication using the Bioruptor Pico sonication device (Diagenode; B01060001) using 20 cycles (30s on and 30s off) at maximum intensity. Purified chromatin was then separated for 1. Immunoprecipitation using 4ug of H3k27ac antibodies (Abcam; ab4729) per ChIP experiment or using 4ug of YY1 antibodies (Santa Cruz; sc-281 X). ChIP-seq experiment for YY1 were performed in biological duplicates. Cells were stimulated with estrogen for 45 minutes, upon which maximum ERα-binding to chromatin occurs. Biological replicates showed very high correlation (R2=0.98), thus only consensus loci were kept for further analyses. 2. Non-immunoprecipitated chromatin, used as Input control and 3. Assessment of sonication efficiencies using a 1% agarose gel. Before construction of ChIP-seq libraries (NEB Ultra II kit, see supplementary methods), enrichment of the immunoprecipitated sample was ascertained using positive and negative controls for ChIP-qPCR. Library preparation was performed using 10 – 50 ng of immunoprecipitated and Input samples. Before sequencing, libraries were again re-tested to confirm enrichment using positive and negative controls.
Patten D.K., Corleone G., Győrffy B., Perone Y., Slaven N., Barozzi I., Erdős E., Saiakhova A., Goddard K., Vingiani A., Shousha S., Pongor L.S., Hadjiminas D.J., Schiavon G., Barry P., Palmieri C., Coombes R.C., Scacheri P., Pruneri G, & Magnani L. (2018). Enhancer mapping uncovers phenotypic heterogeneity and evolution in patients with luminal breast cancer. Nature medicine, 24(9), 1469-1480.
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3

Chromatin immunoprecipitation with H3K27ac antibody

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For each sample, cells were trypsinized for 8–10 min, trypsin was quenched with 10 mL of ES media, and 107 cells were obtained. Cells were spun down, washed with 10 mL PBS, fixed for 12 min in a mix of formaldehyde (to a final concentration of 1%) and Fix Buffer (50 mM HEPES pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl), and then quenched by glycine (final concentration of 0.125 M). Cells were incubated on ice, and then spun down at 1000g for 5 min. Nuclei were prepared by consecutive washes with Rinse 1 Buffer (50 mM HEPES pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X100), Rinse 2 Buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl), and Shearing Buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris HCl pH 8). After a final spin down, cells were resuspended in 100 μL of Shearing Buffer and 1× protease inhibitor cocktail (Calbiochem) nanodroplets (generous gift from Samantha Pattenden),40 (link) and sonicated in an E110 Covaris Sonicator for 4 min or until DNA was sheared to between 70bp and 500bp (as confirmed by agarose gel).
After sonication, the rest of the protocol was performed with a ChIP-IT High Sensitivity Kit (Active Motif, 53040), and an H3K27ac antibody was used for the pull down (Abcam, ab4729).
Butler K.V., Chiarella A.M., Jin J, & Hathaway N.A. (2017). Targeted Gene Repression Using Novel Bifunctional Molecules to Harness Endogenous Histone Deacetylation Activity. ACS synthetic biology, 7(1), 38-45.
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4

Chromatin Immunoprecipitation of Epigenetic Marks

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Chromatin immunoprecipitation was performed as previously described.20 (link) Cerebral cortex was dissected on ice in PBS from DNMT3A mutants and their WT littermates at 8-weeks old and flash-frozen in liquid nitrogen prior to storage at −80°C. Chromatin were fragmented with the Covaris E220 sonicator (5% Duty Factory, 140 Peak Incidence Power, 200 cycles per burst, milliTUBE 1mL AFA Fiber). ChIP was performed with H3K27ac antibody (0.1μg; Abcam, ab4729; n = 2/sex/genotype/mutation; a total of 4 WT and 4 mutants in P900L litters, and a total of 4 WT and 4 mutants in R878H litters) or a MeCP2 antibody (5μL serum per IP; described in Chen et al.,61 (link); n = 2 per genotype), and libraries were generated using Accel-NGS 2S Plus DNA Library Kit (Swift Biosciences). Pooled libraries were sequenced using a NovaSeq 6000 with the Genome Technology Access Center at Washington University in St. Louis, typically yielding 20–50 million (average: 34 million) single-end reads per sample.
Beard D.C., Zhang X., Wu D.Y., Martin J.R., Erickson A., Boua J.V., Hamagami N., Swift R.G., McCullough K.B., Ge X., Bell-Hensley A., Zheng H., Palmer C.W., Fuhler N.A., Lawrence A.B., Hill C.A., Papouin T., Noguchi K.K., McAlinden A., Garbow J.R., Dougherty J.D., Maloney S.E, & Gabel H.W. (2023). Distinct disease mutations in DNMT3A result in a spectrum of behavioral, epigenetic, and transcriptional deficits. Cell reports, 42(11), 113411.
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5

Chromatin Immunoprecipitation of Histone Marks

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Neuro 2a cells were grown to confluency on 150-mm culture dishes. Cells were crosslinked directly on the dishes for 10 min at room temperature with 1% formaldehyde, followed by quenching with 0.125 M glycine for 5 min. Cells were scraped, pelleted, and lysed in cell lysis buffer for 10 min on ice. Nuclei were collected and lysed in 10 mM Tris (pH 8.0), 1% SDS, 1 mM EDTA, and 1 mM EGTA. DNA shearing was performed on a Bioruptor instrument. Chromatin was precleared with Protein A Dynabeads (Invitrogen) for 2 h, and an aliquot was saved as input. Immunoprecipitation was performed using 32 µL Protein A Dynabeads and 5 µL of H3K4me3 antibody (Millipore; 07-473) or 10 µg H3K27ac antibody (Abcam; ab4729). Chromatin was eluted with elution buffer and reverse crosslinked overnight at 65°C, followed by treatment with RNase A for 30 min at 42°C and proteinase K for 3 h at 55°C. DNA was extracted twice with phenol/chloroform and once with chloroform and ethanol precipitated; 10 ng of ChIP DNA was used for library preparation.
Zhao Y.T., Kwon D.Y., Johnson B.S., Fasolino M., Lamonica J.M., Kim Y.J., Zhao B.S., He C., Vahedi G., Kim T.H, & Zhou Z. (2018). Long genes linked to autism spectrum disorders harbor broad enhancer-like chromatin domains. Genome Research, 28(7), 933-942.
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6

Chromatin Immunoprecipitation for Histone Modifications

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Crosslinked shoot and immature ear were subjected to nuclei extraction, chromatin sonication, and immunoprecipitation with H3K4me3 antibody (1 μg/μl, Abcam, cat. no. ab8580) and H3K27ac antibody (1 μg/μl, Abcam, cat. no. ab4729), respectively, as reported59 (link). Detailed procedures for constructing ChIP-seq libraries were described in Supplementary Methods.
Li E., Liu H., Huang L., Zhang X., Dong X., Song W., Zhao H, & Lai J. (2019). Long-range interactions between proximal and distal regulatory regions in maize. Nature Communications, 10, 2633.
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7

Chromatin Immunoprecipitation Sequencing Protocol

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Formaldehyde-fixed cell pellets were snap-frozen in liquid nitrogen and stored at −80 °C before the ChIP assay. Frozen cells were lysed in 120 µl of lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 1 mM phenyl methane sulfonylfluoride, 20 mg/ml sodium butyrate, proteinase inhibitor cocktail; Sigma), and chromatin was sheared by sonication, using a BioRuptor (Diagenode), to generate 100- to 500-bp DNA fragments. Chromatin was diluted in 1 ml of radioimmunoprecipitation assay (RIPA) buffer and immunoprecipitation was done overnight at 4 °C by incubating 1 µl of H3K27ac antibody (rabbit polyclonal IgG, lot# Gr167929-1, Abcam) pre-coated on 10 µl protein A–coated magnetic beads (Invitrogen). Immunocomplexes were captured and washed, and ChIP DNA was eluted as described previously9 (link). 1 ng of ChIP DNA was PCR amplified for 18–22 cycles using whole-genome amplification (WGA) primers (WGA-SEQX, Sigma) following the manufacturer’s instructions. Approximately 100 ng amplified DNA was used for preparing a standard Illumina sequencing library (TruSeq Nano DNA HT Sample Preparation Kit, Illumina). Libraries were sequenced on the HiSeq2500 Illumina platform to obtain 50-bp single-end reads (TruSeq Rapid Kit, Illumina). Samples that failed quality control steps, as previously described9 (link), were eliminated from further downstream steps and analysis.
Engel I., Seumois G., Chavez L., Samaniego-Castruita D., White B., Chawla A., Mock D., Vijayanand P, & Kronenberg M. (2016). Innate-like functions of natural killer T cell subsets result from highly divergent gene programs. Nature immunology, 17(6), 728-739.
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8

Chromatin Immunoprecipitation Protocol

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Detailed method is provided in Supplementary Methods. For immunoprecipitation, Oct4 antibody (Cell Signaling, 5677S) and SRF antibody (Active Motif, 61385, Santa Cruz, sc-335) were used at 6 μg/ChIP sample, H3K4me3 antibody (Millipore, 07-473), H3K27ac antibody (Abcam, Ab4729), and H3K27me3 antibody (Millipore, 07-449) were used at 1 μg/ChIP sample.
Hu X., Liu Z.Z., Chen X., Schulz V.P., Kumar A., Hartman A.A., Weinstein J., Johnston J.F., Rodriguez E.C., Eastman A.E., Cheng J., Min L., Zhong M., Carroll C., Gallagher P.G., Lu J., Schwartz M., King M.C., Krause D.S, & Guo S. (2019). MKL1-actin pathway restricts chromatin accessibility and prevents mature pluripotency activation. Nature Communications, 10, 1695.
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9

ChIP-qPCR Assay for p63 Regulation

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ChIP-qPCR assays were performed on murine embryonic fibroblasts isolated from p63 flox/flox (f/f) mice (gift of Dr. Elsa Flores; Moffitt Cancer Center & Research Institute), as previously described8 (link). Briefly, cells were seeded onto 150 mm dishes at density of 106 cells/dish, and treated 24 h later with adenoviral vectors expressing GFP or Cre recombinase (AdGFP and AdCre; 150 moi). These cells were collected seven days later and processed using ChromaFlash Chromatin Extraction kits (p-2001; Epigenetek) per manufacturer’s protocol. ChIP was then conducted using H3K27Ac antibody (ab4729; Abcam), HDAC1 antibody (815104; Biolegend) or normal rabbit immunoglobulin G (AB-105-C; R&D systems), as previously described27 (link). ChIP-qPCR was performed using primers synthesized by Sigma Aldrich (Supplemental Table S2). Fold-enrichment of PCR products was calculated after normalization with input of all three types of infected cells.
Pinnamaneni J.P., Singh V.P., Kim M.B., Ryan C.T., Pugazenthi A., Sanagasetti D., Mathison M., Yang J, & Rosengart T.K. (2022). p63 silencing induces epigenetic modulation to enhance human cardiac fibroblast to cardiomyocyte-like differentiation. Scientific Reports, 12, 11416.
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10

HiChIP Protocol for Chromatin Interactions

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HiChIP was performed as previously described with modifications [67 (link)]. Briefly, the biotinylated DNA fragments were processed using the ChIP protocol. After sonication, the DNA fragments were incubated with H3K27ac antibody (Abcam, 177178). The binding DNA was treated with proteinase K and purified using EpiTM DNA Clean Beads (Epibiotek, R1809). The selected DNA fragments were applied to generate cDNA libraries using the QIAseq Ultralow Input Library Kit (QIAGEN), according to the manufacturer’s protocol. The libraries were quantified and sequenced on an Illumina NovaSeq 6000 platform.
Jia Q., Tan Y., Li Y., Wu Y., Wang J, & Tang F. (2023). JUN-induced super-enhancer RNA forms R-loop to promote nasopharyngeal carcinoma metastasis. Cell Death & Disease, 14(7), 459.
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