Acetyl histone h3

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Acetyl-Histone H3 is a laboratory reagent used in various biochemical and molecular biology applications. It is a modified form of the histone protein H3, where the lysine residues have been acetylated. Acetylation of histones is a common epigenetic modification that can influence gene expression and chromatin structure.

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6 protocols using acetyl histone h3

Quantifying Histone Acetylation and TGF-β Signaling

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Western blot was performed following our routine method [18 (link)]. The primary antibodies utilized in this work included these for acetyl-histone H4 (06-866, Millipore, Burlington, MA, USA), acetyl-histone H3 (06-599, Burlington, MA, USA), SMAD7 (sc-11392, Santa Cruz, Dallas, TX, USA), and TGFB1 (sc-146, Santa Cruz, Dallas, TX, USA). The applied secondary antibodies were goat anti-rabbit (sc-2004, Santa Cruz, Dallas, TX, USA) and donkey anti-goat (sc-2020, Santa Cruz, Dallas, TX, USA). The blot signals were visualized using Clarity Western ECL Substrate (Bio-Rad, Feldkirchen, Germany) and acquired by ChemiDoc XRS system (Bio-Rad, Feldkirchen, Germany). Coomassie-Brilliant Blue staining was used as a loading control.

Zhang R., Neuhoff C., Yang Q., Cinar M.U., Uddin M.J., Tholen E., Schellander K, & Tesfaye D. (2022). Sulforaphane Enhanced Proliferation of Porcine Satellite Cells via Epigenetic Augmentation of SMAD7. Animals : an Open Access Journal from MDPI, 12(11), 1365.

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Chromatin Immunoprecipitation and Immunoblotting

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Chromatin immunoprecipitation (ChIP) was performed using the following antibodies from Millipore: Histone H3, clone A3S (05-928), Histone H4, Clone 62-141-13 (05-858), acetyl-Histone H4 (06-866), acetyl-Histone H3 (06-599), and trimethyl-Histone H3 (Lys4) (07-473); and from Santa Cruz Biotechnology, anti-ER (sc-8002X, lot K1809). For immunoblotting, primary antibodies were ER (sc-8002X) and GAPDH (sc-25778) and secondary antibodies were goat-anti-rabbit (sc-2004) or donkey anti-mouse-horseradish peroxidase (sc-2020) (all from Santa Cruz Biotechnology).

Khair L., Guikema J.E., Linehan E.K., Ucher A.J., Leus N.G., Ogilvie C., Lou Z., Schrader C.E, & Stavnezer J. (2014). ATM increases AID activity at downstream S regions during class switch recombination. Journal of immunology (Baltimore, Md. : 1950), 192(10), 4887-4896.

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ChIP-seq Analysis of Smek1/2 and Mbd3 in NPCs

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For the ChIP assay, NPCs derived from wild-type or Smek1/2 dKO E11.5 forebrain or transfected with 4 μg pUltra-hot-Mbd3-flag or a pUltra-hot-empty vector for Mbd3 gain-of-function experiments and with pLKO3G-shMbd3 or pLKO3G-shScramble for Mbd3 loss-of-function were treated with 1% formaldehyde for 10 min at room temperature and quenched with 0.125 M glycine for ten more minutes at room temperature. Cross-linked chromatin was sonicated to fragment DNA to 200–1,000 base pairs, and then immunoprecipitation was performed with rabbit anti-IgG, anti-Smek1 (Sigma), anti-Mbd3 (Cell Signaling), anti-HDAC1, anti-HDAC2, anti-MTA1, and acetyl histone H3 (Santa Cruz Biotechnology) antibodies overnight at 4°C, followed by incubation with 50 μl of magnetic Protein A/G Dynabeads (EMD Millipore). Abundance of sequences in immunoprecipitates was determined by PCR and normalized as a fold-value relative to input chromatin. Smek ChIP-seq data were analyzed with the MACS online tool, and cis-regulatory sequences were analyzed using the Genomic Regions Enrichment of Annotations Tool (GREAT) interface (http://bejerano.stanford.edu/great/public/html/). We also utilized the Intergrative Genomics Viewer (IGV v2.3) to visualize distribution of ChIP-seq–identified peaks in different genomic regions. Primer sets for ChIP-qPCR are listed in S6 Table.

Moon B.S., Yun H.M., Chang W.H., Steele B.H., Cai M., Choi S.H, & Lu W. (2017). Smek promotes corticogenesis through regulating Mbd3’s stability and Mbd3/NuRD complex recruitment to genes associated with neurogenesis. PLoS Biology, 15(5), e2001220.

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Chromatin Immunoprecipitation Assay for NF-κB

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The SimpleChIP Enzymatic Chromatin IP Kit (magnetic beads) was purchased from Cell Signaling Technology, and assays were performed according to the manufacturer’s instructions. Antibodies for RelA (NF-kB p65 #8242S CST), p50 (NF-kBp105/50 #13586S), RelB (#10544 CST), p52 (NF-kB p100/52 #37359S CST), Acetyl-Histone H3 (K27, #4353S CST), and SP3 (sc-365220 Santa Cruz). Genome browser (https://genome.ucsc.edu/index.html (accessed on 1 August 2019)) and Jasper (http://jaspar.genereg.net/ (accessed on 1 August 2019)) were used to evaluate DNA sequences for transcription factor binding sites. Analysis of ChIP was performed on a ViiA7 Real-time PCR System (Applied Biosystems) using QuantiTect SYBR Green PCR Kit (Qiagen). The quantification of transcription factors binding to target sites was calculated by measuring the ddCT (Delta-Delta-cycle threshold) ratio of chromatin immunoprecipitation (ChIP) to 2% Input, and the normal rabbit IgG antibody served as a negative control. All primers used for ChIP PCRs are listed in Supplementary Table S2.

Shibuya Y., Kudo K., Zeligs K.P., Anderson D., Hernandez L., Ning F., Cole C.B., Fergusson M., Kedei N., Lyons J., Taylor J., Korrapati S, & Annunziata C.M. (2023). SMAC Mimetics Synergistically Cooperate with HDAC Inhibitors Enhancing TNF-α Autocrine Signaling. Cancers, 15(4), 1315.

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Protein Expression Analysis Protocol

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For analysis of soluble proteins, cells or tissues were homogenized in RIPA lysis buffer, and the total protein content was extracted as detailed in a previously described protocol (Wang et al, 2012). The extracts were analyzed by 8–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by blotting with the following antibodies: E2F1 (Abcam, ab137415, 1:1,000), Cyclin A (Santa Cruz, H‐432, 1:2,000), PCNA (Abcam, ab15497, 1:1,000), P21 (Abcam, ab7960, 1:2,000), TDP‐43 (Proteintech, 10782‐2‐AP; Rosemont, IL, 1:1,000), HDAC1 (Thermo, PA1‐860; Waltham, MA, 1:1,000), γH2AX (Millipore, 05‐636, 1:800), acetyl‐histone H3 (Santa Cruz, SC‐8655, 1:500), total histone H3 (Cell Signaling, #4499, 1:1,000), and tubulin (Abcam, ab4074, 1:5,000). After primary antibody binding, the blots were incubated at room temperature with the appropriate secondary antibodies and Western Lightning Plus‐ECL (PerkinElmer; Waltham, MA). All cell data are repeated at least 3 independent experiments. For quantitative analysis, relative intensities of the bands were normalized against those of internal controls and expressed as mean ± SEM.

Wu C., Jin L., Wang I., Wei W., Ho P., Liu Y, & Tsai K. (2020). HDAC1 dysregulation induces aberrant cell cycle and DNA damage in progress of TDP‐43 proteinopathies. EMBO Molecular Medicine, 12(6), e10622.

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Western Blot Analysis of Cellular Proteins

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Whole-cell extracts were prepared by lysis with 2X Laemmli sample buffer (BioRad, Cat# 1610737) followed by heating at 100 °C for 5 min. Protein concentrations were measured by Pierce 660 nm protein assay reagent (Thermo Fisher, cat#1861426) in the presence of IDCR reagent (Thermo Fisher, cat#22663) according to manufacturer's protocol. About 20 μg of proteins was loaded onto 4–20% Tris-glycine polyacrylamide gels and Western analyses were carried out using standard procedures. Antibodies were obtained from Cell Signaling Technology (pSTAT3^Y705, #9145; STAT3, #4904; SOX2, #3579; Acetyl-Histone H3 (K27), #8173; Histone H3, #3638; Histone H4, #2935; Anti-mouse IgG, HRP linked, #7076; Anti-Rabbit IgG, HRP linked, #7074), MilliporeSigma (Acetyl-Histone H3, #06-599; Acetyl-Histone H4, #06-866), Santa Cruz Biotechnology (CEBPD, #sc-135733; GAPDH, #sc-47724), MBL International Corp (LC3, #PM036), DSHB (α−tubulin, #12G10), and Abcam (Actin, #ab6276).

Poria D.K., Sheshadri N., Balamurugan K., Sharan S, & Sterneck E. (2020). The STAT3 inhibitor Stattic acts independently of STAT3 to decrease histone acetylation and modulate gene expression. The Journal of Biological Chemistry, 296, 100220.

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