Chromatin Immunoprecipitation was performed with anti-H3 antibodies (Abcam, Cambridge, United Kingdom, ab1791), anti-H3K9ac antibodies (Abcam, Cambridge, United Kingdom, ab12179), anti-H3K9me2 antibodies (Abcam, Cambridge, United Kingdom, ab1220), anti-H4K5ac antibodies (Abcam, Cambridge, United Kingdom, ab51997), anti-DNA-RNA Hybrid antibodies (Kerafast, Boston, MA, United States), following the procedure used by Cong et al. (2012) (link). Rabbit serum was used as a negative control for mock immunoprecipitation. Precipitated genomic DNA was subjected to quantitative PCR with primers below that were designed to amplify approximately 200–1000 bp fragments encompassing the promoter region, the exon region and the intergenic spacer (IGS) region of the rDNA gene according to the real-time PCR procedure mentioned above. All primer sequences for ChIP were referenced from published data (Cong et al., 2012 (link)).
Anti h3 antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
The Anti-H3 antibody is a primary antibody that specifically binds to histone H3, a core histone protein found in eukaryotic cells. This antibody can be used in various applications to detect and study the presence and distribution of histone H3 in samples, such as in Western blotting, immunohistochemistry, and chromatin immunoprecipitation (ChIP) experiments.
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Lab products found in correlation
Anti gapdh antibody, by Abcam (3 mentions)
Anti h3k9ac antibody, by Abcam (2 mentions)
Anti h3k9me2 antibody, by Abcam (2 mentions)
Anti h3k9me3, by Abcam (2 mentions)
Ab1791, by Abcam (2 mentions)
Anti h3k27me3 antibody, by Merck Group (2 mentions)
Anti myc antibody, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl method, by Bio-Rad (1 mentions)
Hrp labeled goat anti rabbit igg, by Beyotime (1 mentions)
Horseradish peroxidase hrp labeled goat anti mouse igg, by Beyotime (1 mentions)
Histone extraction kit, by Abcam (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Anti n cadherin, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti phospho egfr 1068, by Cell Signaling Technology (1 mentions)
Anti egfr, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Erlotinib, by Selleck Chemicals (1 mentions)
24 protocols using anti h3 antibody
1
Chromatin Immunoprecipitation of Histone Modifications
2
Whole-cell Protein Extraction and Immunoblotting
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Whole-cell protein extracts were prepared from HeLa cells lysed in extraction buffer (100 mM Tris–HCl pH 7.4, 50 mM NaCl, 5 mM EDTA, and 1 mM PMSF). Proteins were loaded onto a 12% SDS-PAGE gel and separated by electrophoresis. Then, proteins were transferred to PVDF membranes (Millipore, Billerica, MA, United States) and the membranes were incubated with 5% non-fat milk-TBS for 2 h at room temperature. Afterward, the immunoblots were incubated overnight with the following primary antibodies: anti-H3 antibodies (Abcam, Cambridge, United Kingdom, ab1791), anti-H3K9me2 antibodies (Abcam, Cambridge, United Kingdom, ab1220), anti-GAPDH antibodies (Beyotime, Shanghai, China), and anti-RNase H1 monoclonal antibodies (Abcam, Cambridge, MA, United States, ab56560). The secondary antibodies were horseradish peroxidase (HRP) labeled goat anti-mouse IgG (Beyotime, Shanghai, China, A0126) and HRP labeled goat anti-rabbit IgG (Beyotime, Shanghai, China, A3327). Immunoreactivity was determined using the ECL method (Bio-Rad, Hercules, CA, United States) according to the manufacturer’s instructions.
3
Quantifying Histone Modifications in Arabidopsis
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Total histone proteins were extracted with a Histone Extraction kit (Abcam, Cat# AB113476). Briefly, 100 mg of 12-day-old Arabidopsis seedlings were quickly frozen in liquid nitrogen and ground to a powder with steel beads in 1.5 ml Eppendorf safe-locked tubes by shaking. Then 500 μl of 1× Pre-Lysis Buffer was added and thoroughly mixed with the ground sample. The mixture was centrifuged at 8000 × g for 1 min at 4°C. The pellet was then resuspended with 3 volumes (300 μl) of Lysis Buffer and incubated on ice for 30 min. After centrifugation at 11 000 × g for 5 min at 4°C, the supernatant was transferred into a new tube, and then 0.3 volume of the Balance-DTT Buffer was added quickly. The resulting protein solution was used for western blot assay with anti-H3 antibodies (Abcam, Cat# AB1791), anti-H3K9ac antibodies (Abcam, Cat# AB10812) and anti-H3K18ac antibodies (Millipore Sigma, Cat# 07-354).
4
Investigating Signaling Pathways in EGFR-Mediated Epithelial-Mesenchymal Transition
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The antibodies used were anti‐phospho‐EGFR1068 (Cell Signaling Technology, #3777), anti‐EGFR (Cell Signaling Technology, #4267), anti‐ERK (Cell Signaling Technology, #4695), anti‐phospho‐ERK (Cell Signaling Technology, # 4370), anti‐Snail (Cell Signaling Technology, #3879), anti‐E‐cadherin (Cell Signaling Technology, #14472), anti‐vimentin (Cell Signaling Technology, # 5741), anti‐N‐cadherin (Cell Signaling Technology, #13116), anti‐IL‐1β (Cell Signaling Technology, #12703), anti‐Cit‐H3 (Abcam, ab5103), anti‐H3 antibodies (Abcam, ab1791) and anti‐β‐actin antibody (Abcam, ab8226). The inhibitors erlotinib, SHC772984 and Sivelestat (ONO‐5046) were purchased from Selleck Chemicals (Selleck Chemicals, Houston, TX, USA).
5
Chromatin Remodeling Regulation Protocol
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Anti-H4K12ac and anti-H3 antibodies were purchased from Abcam (Paris, France), and anti-BRD2 was from Bethyl Laboratories (Montgomery, TX). Anti-Tip60 antibody was purchased from Upstate Laboratories (Merck Millipore, Darmstadt, Germany). The myc-Suv39H1 expression vector was a kind gift from T. Jenuwein (Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany; Peters et al., 2001 (link)).
All siRNAs were purchased from Eurogentec (Angers, France). Sequences were as follows:
Control siRNA: ACUCAAACUCACGAAGGAA-dTdT.
Tip60-1: UGAGAUUGAUGGACGGAAA-dTdT.
Tip60-2: UGAAGAACAUUGAGUGUAU-dTdT.
BRD2 siRNA: GACAAAGGAGGAACUGGCUUUGGAG-dTdT.
The following primers were used:
Tip60 mRNA: 5′-GACCCCTTCCTCTTCTACGT-3′ and 5′-CCGGTCTTCCCTTCTACTTT-3′.
p400 mRNA: 5′-GAAGTTGGCTGCTGCTAAGA-3′ and 5′-CCTCTTCTGGAAACTTTCTG-3′.
Suv39H1 mRNA: 5′-GGGTCCACTTGCATGTTGTAA-3′ and 5′-GGCAACATCTCCCACTTTGT-3′.
Major satellites (expression and ChIP): 5′-GACGACTTGAAAAATGACGAAATC-3′ and 5′-CATATTCCAGGTCCTTCAGTGTGC-3′.
β2m mRNA (expression and ChIP): 5′-CCCGTTCTTCAGCATTTGGA-3′ and 5′- CCGAACATACTGAACTGCTACGTAA-3′.
p53 promoter: 5′-CAGAGCAGAAAGGGACTTGG-3′ and 5′-CTTCACTTGGGCCTTCAAAA-3′.
All siRNAs were purchased from Eurogentec (Angers, France). Sequences were as follows:
Control siRNA: ACUCAAACUCACGAAGGAA-dTdT.
Tip60-1: UGAGAUUGAUGGACGGAAA-dTdT.
Tip60-2: UGAAGAACAUUGAGUGUAU-dTdT.
BRD2 siRNA: GACAAAGGAGGAACUGGCUUUGGAG-dTdT.
The following primers were used:
Tip60 mRNA: 5′-GACCCCTTCCTCTTCTACGT-3′ and 5′-CCGGTCTTCCCTTCTACTTT-3′.
p400 mRNA: 5′-GAAGTTGGCTGCTGCTAAGA-3′ and 5′-CCTCTTCTGGAAACTTTCTG-3′.
Suv39H1 mRNA: 5′-GGGTCCACTTGCATGTTGTAA-3′ and 5′-GGCAACATCTCCCACTTTGT-3′.
Major satellites (expression and ChIP): 5′-GACGACTTGAAAAATGACGAAATC-3′ and 5′-CATATTCCAGGTCCTTCAGTGTGC-3′.
β2m mRNA (expression and ChIP): 5′-CCCGTTCTTCAGCATTTGGA-3′ and 5′- CCGAACATACTGAACTGCTACGTAA-3′.
p53 promoter: 5′-CAGAGCAGAAAGGGACTTGG-3′ and 5′-CTTCACTTGGGCCTTCAAAA-3′.
6
Quantitative ChIP-qPCR Protocol
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A detailed protocol for ChIP and quantitative PCR analysis can be found at http://www.mimo.unige.ch/STRUBIN_LABb.htm . Briefly, whole-cell extracts equivalent to about 2 × 108 yeast cells were immunoprecipitated with anti-HA antibodies (2 μl, #16B12, Covance), anti-H3 antibodies (1 μl, #1791, Abcam), anti-acetyl-H3K9 (2 μl, #07-352, Millipore), anti-trimethyl-H3K4 (2 μl, #07-473, Millipore) and anti-trimethyl-H3K36 (2 μl, #9050, Abcam). The recovered DNA and at least two standard dilutions of the input DNA were quantified in duplicate by real-time PCR using the KAPA SYBR FAST qPCR Kit Master Mix (2×) Universal (Kapa Biosystems) and the Biorad CFX96 Real-time PCR System. Sequences of the oligonucleotide primers are available upon request. The immunoprecipitation (IP) value for a given region was calculated as the ratio between the IP signal and the respective input DNA signal to correct for variation between different samples and primer pairs. Quantitative sequential ChIP was performed as previously described (27 (link)). All data are representative of at least two completely independent experiments. Independent biological replicates can be found in Supplementary Material.
7
HIV-1 LTR Chromatin Immunoprecipitation
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JurkatTat LTR‐DsRed cells, clone #11, were treated with 3 μM PH02 and/or 10 nM PEP005, for 24 h, and the cells cross‐linked with 1% formaldehyde for 10 min at RT. The cells were resuspended in cold NP‐40 lysis buffer (0.5% NP‐40, 10 mM Tris–HCl pH 7.8, 3 mM MgCl2) containing freshly added protease inhibitor cocktail (PIC, Sigma‐Aldrich) and spun to collect nuclei. The nuclei were sonicated in buffer containing 10 mM Tris–HCl (pH 7.8), 10 mM EDTA, and 0.5% SDS to obtain DNA fragments of 200–2,000 bp using a Bioruptor sonicator (Diagenode). The chromatin fractions were precleared with protein A/G agarose beads (Millipore) and immune‐precipitated with anti‐H3K9‐me3, anti‐H3K9‐ac, or anti‐H3 antibodies (Abcam) according to the manufacturer's instructions. Cross‐links were reversed by incubating the samples at 68°C for 2 h, and DNA was purified using QIAquick PCR columns and assayed by qPCR using primers specific to the HIV‐1 LTR core promoter, AGTGGCGAGCCCTCAGAT, and AGAGCTCCCAGGCTCAAATC. Results are presented as normalized relative to that for histone H3 immunoprecipitates.
8
Western Blot Antibody Dilutions
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The anti-GFP antibody (a mixture of two mouse anti-GFP monoclonal antibodies (Roche)) was used at a dilution of 1:1000, the anti-H3 antibody (Abcam ab1719) was used at a concentration of 1:5000 and the H2A.Bbd antibody (Millipore polyclonal) at a concentration of 1:1000. HRP-coupled secondary antibodies were used at a concentration of 1:5000 and detection was performed using ECL chemiluminescent reagents (GE Healthcare Life Sciences).
9
Characterization of Alzheimer's Pathology
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Lyso-Tracker Red (1:10,000; DND-99) was purchased from invitrogen; anti-TFEB antibody (1:800; 13,372-1-AP) was purchased from proteintech; anti-H3 antibody (1:1,500; 17,168-1-AP) was purchased from Abcam, USA; mouse anti-Aβ antibody (1:1,000 dilution) was purchased from Abcam, USA; mouse anti-β-actin antibody (1:1,000 dilution) was purchased from Abcam, USA; mouse monoclonal antibodies used were agonist Aβ (1:10,000) and β-actin (1:10,000); rabbit monoclonal antibodies used were against TFEB (1:5,000) and H3 (1:10,000); BCA protein quantification kit was purchased from China Biyuntian Biotechnology Co., Ltd.; Aβ40 and Aβ42 ELISA kits were purchased from CUSABIO, China.
10
Chromatin Immunoprecipitation Assay Protocol
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Log phase cells were cross linked with 1% formaldehyde and standard ChIP assays were performed as described previously32 (link). 50 μl of lysate were used per ChIP assay with the following amounts of antibodies: 3 μl Sir2 (in house/480), 10 μl Net1 (Santa Cruz/sc-27758), 10 μl Cdc14 (Santa Cruz/sc-12045), 2.5 μl K56Ac (in house/665), 3 μl Myc (Roche/9E10). DNA was extracted from the immunoprecipitated chromatin using the fast ChIP method34 (link). For the H3 ChIP, 100 μl of chromatin was diluted with 800 ml of 140 μM lysis buffer35 (link) and incubated overnight along with 1 μg of anti-H3 antibody (Abcam/Ab1791) and 50 ml of 50% protein-A sepharose beads. Chromatin from the H3 ChIP assay was heat denatured at 95 °C for 25 min and purified using the QIAquick PCR Purification Kit (QIAGEN). For HU treatment, log phase cells were first arrested by alpha factor for 4 hours and release in 0.2 M HU. Cells were collected at different time and ChIP was performed using antibody against H3 K56Ac and H3 as described before.
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