ChIP assays were performed essentially as described before22 ,26 (link),33 –44 . In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ~200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-BRG1 (Santa Cruz, sc-10768), anti-Sp1 (Abcam, ab13370), anti-SRF (Cell Signaling Technology, 5147), (Santa Cruz, sc-585), anti-SLUG (Cell Signaling Technology, 9585), anti-ZEB1 (Cell Signaling Technology, 3396), anti-SNAIL (Cell Signaling Technology, 3879), anti-acetyl H3 (Millipore, 06-599), anti-acetyl H4 (Millipore, 06-598), anti-histone H3 (Millipore, 06-755), or pre-immune IgG. For re-ChIP, immune complexes were eluted with the elution buffer (1% SDS, 100 mM NaCO3), diluted with the re-ChIP buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris pH 8.1), and subject to immunoprecipitation with a second antibody of interest.
Anti acetyl h3
Manufactured by Merck Group
Sourced in United States, Canada, Italy
Anti-acetyl H3 is a laboratory reagent used to detect and quantify the acetylation of the histone H3 protein. It is a highly specific antibody that binds to the acetylated form of histone H3, allowing researchers to study epigenetic modifications and their role in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti acetyl h4, by Merck Group (7 mentions)
250 sonicator, by Emerson (5 mentions)
Anti trimethyl h3k4, by Merck Group (5 mentions)
Anti brg1, by Santa Cruz Biotechnology (3 mentions)
Anti srf, by Cell Signaling Technology (2 mentions)
Cfx96 cycler, by Bio-Rad (2 mentions)
Anti h3, by Abcam (2 mentions)
Anti h3, by Merck Group (2 mentions)
Anti zeb1, by Cell Signaling Technology (1 mentions)
Anti histone h3, by Merck Group (1 mentions)
Anti sp1, by Abcam (1 mentions)
Anti snail, by Cell Signaling Technology (1 mentions)
Anti slug, by Cell Signaling Technology (1 mentions)
Sybr qpcr master mix, by Toyobo (1 mentions)
Protein a, by GE Healthcare (1 mentions)
Anti rabbit, by Vector Laboratories (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti h3k36me3, by Abcam (1 mentions)
Anti myc, by BioLegend (1 mentions)
Accel ngs 2s plus dna library kit, by Integrated DNA Technologies (1 mentions)
15 protocols using anti acetyl h3
1
Chromatin Immunoprecipitation (ChIP) Assay
2
ChIP-qPCR for Histone Modifications
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Chromatin immunoprecipitation (ChIP) was carried out as previously described (32 (link)) with the oligonucleotides listed in Supplementary Table S2 . Thus, anti-H3 (1.0 μl; Abcam, 1791), anti-acetyl H4 (1.0 μl; Millipore, 06–598), anti-acetyl H3 (1.0 μl; Millipore, 06-599), or anti-trimethyl H3K4 (0.5 μl; Millipore, 07-473) was bound to Protein A (GE-Healthcare, 17078001) agarose beads. Binding for anti-acetyl H4, anti-acetyl H3 and anti-H3K4me3 or for anti-H3 was done overnight in FA lysis buffer containing 1 M NaCl or in FA lysis buffer with 275 mM NaCl, respectively. Precipitates were washed with the same buffer, once with FA lysis buffer containing 1.5 M NaCl for anti-acetyl H4, anti-acetyl H3 and anti-H3K4me3 or with FA lysis buffer containing 500 mM NaCl for anti-H3, once with 10 mM Tris–HCl (pH 8.0), 0.25 M LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate, and once with TE (10 mM Tris–HCl [pH 8.0], 1 mM EDTA). Precipitated DNAs were analyzed by real-time quantitative PCR using SYBR qPCR Master mix (TOYOBO, QPS-201T) and CFX96 cycler (Bio-Rad).
3
Quantifying Histone Acetylation in Brain
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Bilateral DLS and dHPC were obtained from 1‐mm coronal brain sections 24 hr after probe testing. Western blotting was performed as described previously (Kennedy et al., 2013 ). Briefly, tissue was homogenized in 50–90 μL of 1 M HEPES lysis buffer (1% SDS) containing protease and phosphatase inhibitors using an ultrasonic processor. Protein concentrations were determined using a DC protein assay and 10–20 μg samples of total protein were electrophoresed on 18% Tris‐HCl polyacrylamide gels. Proteins were transferred to a PVDF membrane, blocked for 1 hr in 5% BSA, and incubated overnight at 4°C with either anti‐acetyl H3 (Millipore, Burlington, MA, Cat. # 06‐599, RRID:AB_2115283, 1:5,000) or anti‐GAPDH (Cell Signaling Technology, Danvers, MA, Cat. #2118, RRID:AB_561053, 1:60,000) antibodies. Membranes were then incubated with secondary antibody conjugated to horseradish peroxidase for 1 hr at room temperature (anti‐Rabbit, Vector Laboratories, Burlingame, CA, Cat. # PI‐1000) and bands were visualized using SuperSignal West Dura substrate. Bands were quantified with the NIH ImageJ software and normalized to GAPDH to control for equal loading.
4
Chromatin Immunoprecipitation with Histone Modifications
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Cells fixed with 1% formaldehyde were subjected to ChIP as previously described6 (link) with oligonucleotides as listed in Supplementary Table 2 . The following histone antibodies were used: anti-H3K36me3 (Abcam ab9050), anti-acetyl H4 (Millipore 06–598), anti-acetyl H3 (Millipore 06–599), anti-myc (BioLegend 626802) and anti-H3 (Abcam ab1791). Except for H3 acetylation, all ChIP-seqs included S. pombe “spike-in” added at 10% relative to Saccharomyces cerevisiae chromatin.
Precipitated DNAs were analyzed by quantitative real-time PCR using THUNDERBIRD® SYPR qPCR Mix (TOYOBO) and CFX96 cycler (Bio-Rad). The DNA libraries for ChIP-seq were prepared using Accel-NGS 2 S Plus DNA Library Kit (Swift Biosciences) and sequenced on the HiSeq2500 platform (Illumina) following the manufacturer’s instructions.
Precipitated DNAs were analyzed by quantitative real-time PCR using THUNDERBIRD® SYPR qPCR Mix (TOYOBO) and CFX96 cycler (Bio-Rad). The DNA libraries for ChIP-seq were prepared using Accel-NGS 2 S Plus DNA Library Kit (Swift Biosciences) and sequenced on the HiSeq2500 platform (Illumina) following the manufacturer’s instructions.
5
ChIP Assay Protocol for Epigenetic Profiling
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ChIP assays were performed essentially as described before [[29] , [30] , [31] , [32] , [33] , [34] , [35] (link)]. Briefly, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ~500 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-acetyl H3 (06-599, Millipore), anti-acetyl H4 (06-598, Millipore), anti-acetyl H3K27 (17-683, Millipore), anti-H4K16 (13534, Cell Signaling), anti-p300 (sc-585, Santa Cruz), anti-SRF (5147, Cell Signaling), and anti-KAT8 (13842-1, Proteintech) antibodies. Precipitated genomic DNA was amplified by real-time PCR with primers that span the target promoters or a control promoter (GAPDH). Serially diluted genomic DNA extracted from normal cells/tissues was used to generate a standard curve to calculate the amount of DNA being precipitated by a particular antibody. A total of 10% of the starting material is also included as the input. Data are then normalized to the input and expressed as fold changes compared to the control group. All experiments were repeated at least three times.
6
Histone Extraction and Western Blot Analysis
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Samples were homogenized in Triton extraction buffer (TEB, 0.5 % Triton X 100, 2 mM phenylmethylsulfonyl fluoride (PMSF), 0.02 % NaN3 in PBS) and centrifuged at 2000 rpm for 10 min at 4 °C. Pellets were resuspended in 0.2 N HCl, and histones were acid extracted overnight at 4 °C. Histones were then precipitated in 100 % trichloroacetic acid (Sigma-Aldrich), washed with cold acetone, and allowed to air dry. Pellets were either stored at −20 or dissolved in ddH2O. Histone extracts were loaded onto 12 % SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in 5 % dry milk in TBST and incubated with anti-acetyl-H3K27 (Abcam) 1:2000, anti-acetyl H3 (Millipore) 1:2000, or anti-H3 (Millipore) 1:10000. Horseradish peroxidase-conjugated anti-rabbit (Rockland), 1:10,000, was used as secondary antibodies. Chemiluminescent signals were detected with Clarity Western ECL Detection Kit (Bio-Rad) and imaged using a ChemiDoc MP System (Bio-Rad).
7
Chromatin Immunoprecipitation (ChIP) Assay Protocol
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Chromatin Immunoprecipitation (ChIP) assays were performed essentially as described before (Coarfa et al., 2020 (link); Hu et al., 2020 (link); Jehanno et al., 2020 (link); Maity et al., 2020 (link); Mallik et al., 2020 (link); Moon et al., 2020 (link); Shen et al., 2020 (link); Wang J. N. et al., 2020 (link); Wang S. et al., 2020 (link); Zhang et al., 2020 (link); Zhao et al., 2020 (link); Marti et al., 2021 (link); Peng et al., 2021 (link); Rashid et al., 2021 (link)). In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ∼200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-acetyl H3 (Millipore, 06-599), anti-trimethyl H3K4 (Millipore, 07-473), anti-trimethyl H3K9 (Diagenode, C15410193), anti-trimethyl H3K27 (Millipore, 04-449), anti-trimethyl H4K20 (Millipore, 07-463), anti-KDM4A (Abcam, ab191433), anti-KDM4B (Bethyl Laboratories, A301-477A), anti-KDM4C (Bethyl Laboratories, A300-885A), or anti-KDM4D (Proteintech, 22591-1-AP) antibodies.
8
Chromatin Immunoprecipitation (ChIP) Protocol for Epigenetic Analysis
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Chromatin Immunoprecipitation (ChIP) assays were performed essentially as described before (Wu et al., 2020 (link)). In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and phenylmethylsulfonyl fluoride (PMSF). DNA was fragmented into ∼200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-BRG1 (Abcam, ab110641), anti-E2F5 (Thermo Fisher, PA5-81166), anti-TFDP1 (Thermo Fisher, PA5-86135), anti-acetyl H3 (Millipore, 06–599), anti-trimethyl H3K4 (Millipore, 17–614), anti-RNA polymerase II (Abcam, ab264350), anti-p300 (Abcam, ab275378), anti-KMT2A (Bethyl laboratories, A300-087A), or pre-immune IgG. Precipitated DNA was amplified with the following primers: 5′-AGGCTTTTCCCCGCCCTC-3′ and 5′-AGAGAGAGAGAGTCGGAAAAAAG-3′.
9
ChIP Assay for Hes5, SIRT1, and Histone Modifications
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Chromatin immunoprecipitation (ChIP) assays were performed essentially as described before [57 (link), 73 (link)–90 ]. In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ~200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-Hes5 (Abcam, ab194111), anti-SIRT1 (Santa Cruz, sc-74504), anti-acetyl H3 (Millipore, 06-599), anti-acetyl H4 (Millipore, 06-866), or pre-immune IgG. Precipitated DNA was amplified with the following primers: #1, 5’-AGAGTGAGACAGGGCCAAGAC-3’ and 5’-AAACCGAAATTGCTCAACACAC-3’; #2, 5’-AAACCCACAACGTATTA-3’ and 5’-ATGCAGCAATGAACAAC-3’.
10
Cytokine Quantification and Epigenetic Analysis
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RPMI 1640, fetal bovine serum (FBS) and other cell culture reagents were purchased from Gibco BRL Co. (Grand Island, NY, USA). All primary and secondary antibodies, unless otherwise stated, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-acetyl-H3 and anti-acetyl-H4 antibodies were purchased from Millipore (Bedford, MA, USA). The enzyme-linked immunosorbent assay (ELISA) kits for interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) were purchased from R&D Systems (Minneapolis, MN, USA). Histone extraction kit was obtained from Abcam (Cambridge, UK). Other chemicals, unless stated otherwise, were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
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