Anti h3k4me3

Manufactured by Merck Group

Sourced in United States, United Kingdom, Morocco, Germany

Anti-H3K4me3 is a laboratory reagent used for the detection and analysis of the trimethylation of lysine 4 on histone H3 (H3K4me3), a post-translational modification associated with active gene transcription. This product is designed for use in various research applications, such as chromatin immunoprecipitation (ChIP), Western blotting, and immunofluorescence microscopy.

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Lab products found in correlation

Anti h3k27me3, by Merck Group (38 mentions) Anti h3k27ac, by Abcam (11 mentions) Anti h3k4me2, by Merck Group (11 mentions) Anti h3k4me1, by Merck Group (11 mentions) Anti h3k9ac, by Merck Group (9 mentions) Anti h3k36me3, by Abcam (8 mentions) Anti h3k9me3, by Merck Group (8 mentions) Anti h3, by Abcam (7 mentions) Anti h3k27ac, by Merck Group (7 mentions) Anti h3, by Merck Group (6 mentions) Anti h3k9me2, by Merck Group (6 mentions) Anti rna polymerase 2, by Abcam (5 mentions) Qiaquick pcr purification kit, by Qiagen (5 mentions) Anti h3k14ac, by Merck Group (5 mentions) Anti h3k4me1, by Abcam (5 mentions) Hiseq 2500, by Illumina (4 mentions) Anti h3k9me3, by Abcam (4 mentions) Anti h3k36me3, by Merck Group (4 mentions) Anti h3k27me1, by Merck Group (4 mentions) Anti h3k9me2, by Abcam (4 mentions)

Spelling variants of this product

H3K4me3 (186 citations) Anti-H3K4me3 antibody (26 citations) Rabbit anti-H3K4me3 (11 citations) Rabbit anti-H3K4me3 (07–473), (3 citations) Anti-H3K4me3/H3ac/H3K9ac/H4ac/H4K16ac (2 citations) Anti-H3K4me3 (07-473) (2 citations) Anti-H3K4me3 (17–614) (2 citations)

123 protocols using anti h3k4me3

Oocyte H3K4me3 ChIP-Seq Protocol

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For H3K4me3 ChIP-Seq in oocytes, we isolated SCP3 positive meiotic oocytes from 14 15.5 dpc females using BiTS-CHiP^{42 (link)}. Briefly, this method uses FACS to isolate nuclei on the basis of the presence of an intra-nuclear marker (in this case, anti-SCP3 (Santa Cruz: sc-74569)). Oocytes were isolated by gating for 4C nuclei with SCP3 signal above that from secondary antibodies alone. The gating strategy is outlined in Supplementary Figure 1. Kapa Hyper Prep kit (catalog #KR0961) was used to prepare the sequencing library due to the limited starting material relative to experiments in whole testis.
Testis sample preparation was performed as described previously^{4 (link)}. The following antibodies were used: anti-DMC1: Santa Cruz (C-20, sc-8973), anti-DMC1: (custom), anti-H3K4me3: Millipore (#07–473). All sequencing was performed on an Illumina HiSeq 2500 at the NIDDK genomics core.

Brick K., Thibault-Sennett S., Smagulova F., Lam K.W., Pu Y., Pratto F., Camerini-Otero R.D, & Petukhova G.V. (2018). Extensive sex differences at the initiation of genetic recombination. Nature, 561(7723), 338-342.

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Pregnancy and Lactation Chromatin Profiling

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Frozen-stored mammary tissues harvested at day 13 of pregnancy (p13), p14, p16 and day 1 of lactation (L1) were ground into powder with a mortar and pestle. Chromatin was fixed with 1% formaldehyde at room temperature for 10 min and the fixation was quenched with Glycine at a final concentration of 0.125M. Samples were processed as previously described⁷⁰. The following antibodies were used for ChIP-Seq: anti-STAT5A (Santa Cruz, sc-1081), anti-GR (Thermo Scientific, PA1-511A), anti-NFIB (Santa Cruz, sc-5567), anti-ELF5 (Santa Cruz, sc-9645), anti-MED1 (Bethyl Laboratory, A300-793A), anti-H3K27ac (Abcam, ab4729), anti-H3K4me3 (Millipore, 17-614), and anti-RNA Polymerase II (Abcam, ab5408). Libraries for the next generation sequencing (NGS) were prepared as previously described⁷⁰ and sequenced with HiSeq 2000 (Illumina).

Shin H.Y., Willi M., HyunYoo K., Zeng X., Wang C., Metser G, & Hennighausen L. (2016). Hierarchy within the mammary STAT5-driven Wap super-enhancer. Nature genetics, 48(8), 904-911.

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Chromatin Immunoprecipitation Antibodies

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The antibodies used were anti-H3K4me3(Millipore, Billerica, MA 17–614), anti-H3K9me3(abcam, Cambridge, United Kingdom ab8898), anti-GFP(abcam, Cambridge, United Kingdom ab290), anti-H3(abcam, Cambridge, United Kingdom ab1791), anti-H3K9ac(Wako, Richmond, VA 309–32379).

Wang W., Chaturbedi A., Wang M., An S., Santhi Velayudhan S, & Lee S.S. (2018). SET-9 and SET-26 are H3K4me3 readers and play critical roles in germline development and longevity. eLife, 7, e34970.

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Chromatin Immunoprecipitation (ChIP) Protocol

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Frozen-stored mammary tissues collected from lactation day 1 (L1) were ground into powder with mortar and pestle and then crosslinked with 1% formaldehyde (Sigma–Aldrich) for 10 min. After adding 0.125 M glycine to stop crosslinking, nuclei were isolated with Farnham Lysis Buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, supplemented with PMSF and proteinase inhibitor cocktails). The chromatin was fragmented to 200–500 bp using sonicator 3000 (25 cycles; 30 s pulse/30 s rest, Misonix Sonicators) and further lysed in RIPA buffer. One milligram of chromatin was immunoprecipitated with Dynabeads Protein A (Novex) coated with anti-H3K4me3 (Millipore, 17-614), anti-H3K27ac (Abcam, ab4729), anti-RNA Polymerase II (Abcam, ab5408), anti-MED1 (Bethyl Laboratory, A300-793A), or anti-STAT5A (Santa Cruz, sc-1081). After serial bead washes, ChIP DNA was reverse-crosslinked at 65°C overnight in the presence of 1% SDS and 1 mg/ml of Proteinase K (Roche), and DNA was purified with QIAquick PCR Purification Kit (Qiagen). The DNA fragments were blunt-ended using End-it DNA End-Repair Kit (Epicentre Biotechnology), ligated to the Illumina Indexed DNA adaptors, and sequenced with HiSeq 2000 (Illumina).

Metser G., Shin H.Y., Wang C., Yoo K.H., Oh S., Villarino A.V., O'Shea J.J., Kang K, & Hennighausen L. (2015). An autoregulatory enhancer controls mammary-specific STAT5 functions. Nucleic Acids Research, 44(3), 1052-1063.

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Chromatin Immunoprecipitation (ChIP) Protocol

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ChIP was performed as described previously (18 (link)). Cells were cross-linked for 10 min using 1% formaldehyde. After sonication and pre-clearing, anti-Sp1 (Active-Motif; 39058), anti-H3K4Me3 (Millipore; 04-745), anti-CTCF (Active-Motif; 91285), anti-Pol2 (Millipore; 05-623) and IgG (Santa Cruz Biotechnology) antibodies were used for immunoprecipitation at a concentration of 1 μg per 1 million cells. ChIP eluate was used to perform ChIP-qPCR with specific primers. Primers are shown in Supplementary Table S1.

Akıncılar S.C., Chua J.Y., Ng Q.F., Chan C.H., Eslami-S Z., Chen K., Low J.L., Arumugam S., Aswad L., Chua C., Tan I.B., DasGupta R., Fullwood M.J, & Tergaonkar V. (2022). Identification of mechanism of cancer-cell-specific reactivation of hTERT offers therapeutic opportunities for blocking telomerase specifically in human colorectal cancer. Nucleic Acids Research, 51(1), 1-16.

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Antibody Validation for Epigenetic Analysis

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Antibodies were purchased from the following companies: anti‐H3K27me1, anti‐H3K27me3, anti‐H3K79me1, anti‐H3K79me2, anti‐histone H3, anti‐hnRNP I, anti‐KDM4A, anti‐KDM5A, anti‐KDM5C, anti‐snRNP70, anti‐tubulin, and anti‐U2AF2 from Abcam (Cambridge, MA, USA); anti‐hnRNP A2/B1 from Acris (San Diego, CA, USA); anti‐SR protein‐specific kinases (SRPK)1 and anti‐SRPK2 from BD Biosciences (San Diego, CA, USA); anti‐AKT, anti‐H3K27me2, anti‐protein phosphatase‐1 (PP1), anti‐phospho‐PP1 at Thr320, anti‐phospho‐AKT at Ser473, and anti‐cleaved‐CASPASE‐3 from Cell Signaling Technology (Billerica, MA, USA); anti‐H3K4me1, anti‐H3K4me2, anti‐H3K4me3, and anti‐H3K36me3 from Millipore; anti‐pro‐CASPASE‐3 and anti‐KDM7A from GeneTex (SanAntonio, TX, USA); anti‐hnRNP C1/C2, anti‐BAX, and anti‐BCL2L1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti‐hnRNP A1 from Sigma; and anti‐SRSF1 and anti‐SRSF3 from Zymed (San Francisco, CA, USA).

Lee C., Chang W., Chang Y., Yang J., Chang C., Hsu K., Chen Y., Liu T., Chen Y., Lin S., Wu Y, & Chang J. (2019). Alternative splicing in human cancer cells is modulated by the amiloride derivative 3,5‐diamino‐6‐chloro‐N‐(N‐(2,6‐dichlorobenzoyl)carbamimidoyl)pyrazine‐2‐carboxide. Molecular Oncology, 13(8), 1744-1762.

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Immunofluorescence of Key Cellular Proteins

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Primary antibodies were used at the following dilutions: anti-tubulin (1:15,000, Sigma), anti-H3K4me3 (1:5000, Millipore), anti-H3K4me1 (1:5000, Millipore), anti-RAD51 (1:50, Santa Cruz), anti-PCNA (1:2000, Santa Cruz), anti-total H3 (1:1000 Millipore), and anti-Mre11 (1:10,000, gift from J. Petrini, MSKCC). MEFs were prepared for immunofluorescence by growth on 18 mm × 18 mm glass cover slips. Lymphocytes were dropped onto slides coated with CellTak (BD Biosciences). Cells were fixed with methanol and incubated with primary antibody as indicated.

Chaudhuri A.R., Callen E., Ding X., Gogola E., Duarte A.A., Lee J.E., Wong N., Lafarga V., Calvo J.A., Panzarino N.J., John S., Day A., Crespo A.V., Shen B., Starnes L.M., de Ruiter J.R., Daniel J.A., Konstantinopoulos P.A., Cortez D., Cantor S.B., Fernandez-Capetillo O., Ge K., Jonkers J., Rottenberg S., Sharan S.K, & Nussenzweig A. (2016). Replication Fork Stability Confers Chemoresistance in BRCA-deficient Cells. Nature, 535(7612), 382-387.

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Chromatin Immunoprecipitation (ChIP) Assay

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Cells were grown to 80% confluence, washed twice in warm PBS, dissociated with 0.5x Accutase (Catalogue # SCR005; Millipore), and re-suspended in warm medium (DMEM F-12 Catalogue # 11320-033; Invitrogen) containing 0.1 volume crosslinking solution (Kondo, Shen, Yan, Huang & Issa, 2004 (link)). ChIP reactions were performed as described previously (Veazey et al., 2015 (link)) followed by DNA purification with a Qiaquick PCR Cleanup kit (Catalogue # 28106; QIAGEN). Antibodies used include: anti-H3K4me3 (Catalogue # 04-745; Millipore), anti- H3K27me3 (Catalogue # 39155; Active Motif), anti-H3K9ac (Catalogue # 07-352; Millipore), and anti-H3K9me2 (Catalogue # 39239; Active Motif). Antibodies for modified histones were used at 1 μg/ChIP reaction. The concentration of IgG (Catalogue # SC-2027; Santa Cruz) was also used at 1 ug/ChIP reaction. For analysis of candidate loci, real-time PCR was performed with the Dynamo Flash supermix (Catalogue # F-415XL; Thermo Scientific) according to the recommended protocol. Reactions were performed on a Bio-Rad CFX384 Touch PCR system. Data was analyzed using the formula previously described (Mukhopadhyay, Deplancke, Walhout & Tissenbaum, 2008 (link)). Primer sequences are listed in Table S1- Primer Sequences.

Veazey K.J., Wang H., Behdi Y.S., Skiles W.M., Chang R.C, & Golding M.C. (2017). Disconnect between alcohol-induced alterations in chromatin structure and gene transcription in a mouse embryonic stem cell model of exposure. Alcohol (Fayetteville, N.Y.), 60, 121-133.

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ChIP-seq Analysis of H3K27me3 and H3K4me3

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ChIP-seq of trifoliate leaves of W05 was performed following the procedures of a previous study with minor modifications [24 (link)]. ChIP-seq grade anti-H3K27me3 (Diagenode, C15410195, Denville, NJ, USA) and anti-H3K4me3 (Millipore, 07473, Burlington, VT, USA) antibodies were used for chromatin immunoprecipitation. About 5 ng ChIP’ed DNA or input DNA was used for ChIP-seq library construction via TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501, Nanjing, China). ChIP-seq libraries were sequenced in PE150 mode of the Illumina X Ten platform. Adaptor trimming, low-quality reads removing, and mapping were performed using TrimGalore and Bowtie2. The mapped reads (>MAPQ30) were used for peak-calling using MACS2 [25 (link)], and the parameters were set as ‘—trackline —extsize 147 —broad -q 0.01 —nomodel -g 1.0e + 9 —buffer-size 500,000’. An input library was used as control.

Huang M.K., Zhang L., Zhou L.M., Yung W.S., Li M.W, & Lam H.M. (2021). Genomic Features of Open Chromatin Regions (OCRs) in Wild Soybean and Their Effects on Gene Expressions. Genes, 12(5), 640.

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Chromatin Immunoprecipitation and qPCR Analysis

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Cells were grown to 80% confluence, washed twice in warm PBS, trypsinized, and re-suspended in warm media containing 0.1 volume crosslinking solution (Kondo et al., 2004). ChIP reactions were performed as described previously (Martens et al., 2005) followed by DNA purification with a Qiaquick PCR Cleanup kit (Cat# 28106; Qiagen). Antibodies used include: anti-H4R3me2s (Cat# ab5823; Abcam), anti-H3K4me3 (Cat# 04-745; Millipore), anti-H3K27me3 (Cat# 07-449; Millipore), and anti-p53 (Cat#2524, Cell Signaling Technology). Antibodies for modified histones and p53 were added at 1ug to 5ug/ChIP reaction. The concentration of IgG (Cat# 12-370; Millipore) was adjusted from 1μg to 5μg as appropriate. For qPCR analysis, real-time PCR was performed with the iTaq SYBRgreen Supermix (Cat# 172-5121; Biorad) according to manufacturer’s protocol. Primer sequences were derived from the enhancer and promoter regions of CDKN1a. Reactions were performed on an ABI 7900HT Fast Real-Time PCR system.

Kaushik S., Liu F., Veazey K., Gao G., Das P., Neves L., Li K., Zhong Y., Lu Y., Giuliani V., Bedford M.T., Nimer S.D, & Santos M.A. (2017). Genetic deletion or small molecule inhibition of the arginine methyltransferase PRMT5 exhibit anti-tumoral activity in mouse models of MLL-rearranged AML. Leukemia, 32(2), 499-509.

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