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Anti h3k4me3 antibody

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The Anti-H3K4me3 antibody is a laboratory reagent designed to detect the trimethylation of lysine 4 on histone H3. This post-translational modification is associated with active gene transcription and is a common epigenetic marker. The antibody can be used in various applications, such as chromatin immunoprecipitation (ChIP) and Western blotting, to study the distribution and dynamics of this histone modification.

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Lab products found in correlation

Bioruptor pico, by Diagenode (1 mentions) Anti mll1 antibody, by Active Motif (1 mentions) Ab171870, by Abcam (1 mentions) Fugene hd transfection reagent, by Promega (1 mentions) Anti nf κb p65 antibody, by Santa Cruz Biotechnology (1 mentions) S aureus, by American Type Culture Collection (1 mentions) Unc1999, by Merck Group (1 mentions) Anti h3k27ac antibody, by Diagenode (1 mentions) 2 deoxy d glucose 2 dg, by Merck Group (1 mentions) Anti h3k9me3 antibody, by Active Motif (1 mentions) Trypsin gold, by Promega (1 mentions) Protein g dynabead, by Thermo Fisher Scientific (1 mentions) Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions) Anti histone h3 antibody, by Abcam (1 mentions) Anti m6a antibody, by Synaptic Systems (1 mentions) Anti β actin antibody, by Cell Signaling Technology (1 mentions) Anti mettl3 antibody, by Abcam (1 mentions) Anti ythdf2 antibody, by Proteintech (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

H3K4me3 (62 citations) Anti-H3K4me3 (33 citations) H3K4me3 antibody (10 citations)

9 protocols using anti h3k4me3 antibody

1

ChIP-qPCR Analysis of H3K4me3 and MLL1

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As previously described (48 (link)), cells were fixed in 1% paraformaldehyde and glycine. This was followed by lysis and sonication using a Bioruptor Pico (Diagenode) to generate 300–500 bp fragments. Samples were then incubated overnight with anti-H3K4me3 antibody (39159, Active Motif), anti-MLL1 antibody (61296, Active Motif), or isotype control (rabbit polyclonal IgG; ab171870, Abcam), followed by the addition of protein A or protein G Sepharose beads (Thermo Fisher Scientific). Beads were washed; then, DNA was eluted and purified using phenol/chloroform/isoamyl alcohol extraction. The DNA was then precipitated using ethanol. H3K4me3 deposition was measured by qPCR using 2× SYBR PCR mix (Invitrogen), and primers (forward, 5′-TCTCTGACACCTTTGCCTGA-3′; reverse, 5′-CCAGTTCTCTTCCCTTGCAC-3′) were designed to flank the Ifnk promoter using NCBI Primer-BLAST.
Wolf S.J., Audu C.O., Joshi A., denDekker A., Melvin W.J., Davis F.M., Xing X., Wasikowski R., Tsoi L.C., Kunkel S.L., Gudjonsson J.E., O’Riordan M.X., Kahlenberg J.M, & Gallagher K.A. (2022). IFN-κ is critical for normal wound repair and is decreased in diabetic wounds. JCI Insight, 7(9), e152765.
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2

Visualizing KDM5A and H3K4me3 in HEK293 Cells

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HEK293 cells (2 × 105 cells/well) were plated on glass coverslips in 12-well plates and cultured for 2 days. Cells were transfected with Flag-KDM5A plasmid or empty vector using FuGENE HD Transfection Reagent (Promega). After 24 h, culture media were changed and inhibitors (2 μM) or DMSO (0.1%) were added. After 2 days, coverslips were washed with PBS and the cells were fixed in 70% methanol, followed by blocking for 30 min with 20% (v/v) Blocking One (Nacalai)/Milli-Q water. Cells were then incubated for 60 min with primary antibodies (anti-Flag antibody (M185-3S; MBL Inc.; 1:3,000 dilution) and anti-H3K4me3 antibody (39915; Active Motif Inc.; 1:1500 dilution). The cells were washed with PBS and incubated for 60 min with secondary antibodies as follows: anti-mouse IgG conjugated Alexa488 (A11029; Cell Signaling Technology Inc.; 1:1,000 dilution) to detect Flag and anti-rabbit IgG conjugated CF555 (20033; Biotium Inc.; 1:1,000 dilution) to detect H3K4me3. After washing with PBS, the cells were stained with DAPI and mounted for viewing.
Mitsui E., Yoshida S., Shinoda Y., Matsumori Y., Tsujii H., Tsuchida M., Wada S., Hasegawa M., Ito A., Mino K., Onuki T., Yoshida M., Sasaki R, & Mizukami T. (2019). Identification of ryuvidine as a KDM5A inhibitor. Scientific Reports, 9, 9952.
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3

Profiling Histone H3K4me3 in FFPE Tissues

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At the end of dietary treatment, mice were sacrificed by cervical dislocation, and livers were washed in phosphate buffer (PBS) and incubated overnight at room temperature (RT) in 4% formalin solution. Formalin-fixed samples were then dehydrated by increasing the concentration of ethanol (from 70% to 100%) and subsequently embedded in paraffin.
Chromatin extraction was performed on four sections of formalin-fixed, paraffin-embedded (FFPE) tissues following the PAT-ChIP procedure [30 (link)]. Extracted chromatin was immunoselected with the anti-H3K4me3 antibody (Active Motif, Carlsbad, CA USA, #39159). The bound fractions were decrosslinked, and purified DNA was used for library preparation. Libraries were then sequenced in 50 bp single-read mode on a HiSeq 2000 sequencer.
Persico G., Casciaro F., Marinelli A., Tonelli C., Petroni K, & Giorgio M. (2021). Comparative Analysis of Histone H3K4me3 Distribution in Mouse Liver in Different Diets Reveals the Epigenetic Efficacy of Cyanidin-3-O-glucoside Dietary Intake. International Journal of Molecular Sciences, 22(12), 6503.
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4

Immunomodulatory Effects of Epigenetic Agents

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The following are the reagents, antibodies, and bacteria used in the experiment: BLP (EMC Microcollections, Tubingen, Germany), BCG (Chengdu Institute Biological Products Co. Ltd, Chengdu, China), UNC1999 (Sigma-Aldrich, St. Louis, MO), C646 (Sigma-Aldrich), 2-deoxy-D-glucose (2-DG) (Sigma-Aldrich), anti-NF-κB p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-H3K4me3 antibody (Active Motif, Carlsbad, CA), anti-H3K27Ac antibody (Diagenode, Denville, NJ), anti-H3K9me3 antibody (Active Motif), S. aureus (American Type Culture Collection, Manassas, VA), and Salmonella typhimurium (S. typhimurium) (American Type Culture Collection).
Zhou H., Lu X., Huang J., Jordan P., Ma S., Xu L., Hu F., Gui H., Zhao H., Bai Z., Redmond H.P., Wang J.H, & Wang J. (2022). Induction of Trained Immunity Protects Neonatal Mice Against Microbial Sepsis by Boosting Both the Inflammatory Response and Antimicrobial Activity. Journal of Inflammation Research, 15, 3829-3845.
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5

Chromatin Immunoprecipitation and Mass Spectrometry

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Nuclei were isolated from ∼5 million ES cells under hypotonic conditions and the samples were incubated overnight at 4°C with an anti-H3K4me3 antibody (#39159; Active Motif) in the presence of low-salt binding buffer (150 mM NaCl, 50 mM Tris–HCl, pH 8.0, and 1% NP-40), protease inhibitors, and Protein G Dynabeads (Invitrogen). The following day, after several rounds of bead washing with binding buffer, the samples were incubated overnight at 37°C with Tris, pH 8.8, and 300 ng Trypsin Gold (Promega). In total, four samples were prepared for each condition (control and Ssrp1 KD). For the full protein interactome of both FACT subunits, nuclei were extracted as descripted above, and anti-Ssrp1 and anti-Supt16 antibodies were used. Peptides were desalted using StageTips (Rappsilber et al, 2003 (link)) and dried. The peptides were resuspended in 0.1% formic acid and analyzed using liquid chromatography—mass spectrometry (LC-MS/MS).
Mylonas C, & Tessarz P. (2018). Transcriptional repression by FACT is linked to regulation of chromatin accessibility at the promoter of ES cells. Life Science Alliance, 1(3), e201800085.
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6

Protein Expression and Modification Analysis

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Western blotting was performed as described previously [30 (link)]. The anti-METTL3 antibody (#ab195352) and anti-Ki67 (#ab156956) were purchased from Abcam. The anti-IFIT2 antibody (#DF8962) was purchased from Affbiotech. The anti-H3K4me3 antibody (#39160) was purchased from Active Motif. The anti-β-actin antibody (#4970) and anti-rabbit IgG, HRP-linked antibody (#7071) was purchased from Cell Signaling Technology. The anti-m6A antibody (#202003) was purchased from Synaptic Systems. The anti-histone H3 antibody (#ab21054) was purchased from Abcam. The anti-YTHDF2 antibody (#24744-1-AP) was purchased from Proteintech.
Xu Q.C., Tien Y.C., Shi Y.H., Chen S., Zhu Y.Q., Huang X.T., Huang C.S., Zhao W, & Yin X.Y. (2022). METTL3 promotes intrahepatic cholangiocarcinoma progression by regulating IFIT2 expression in an m6A-YTHDF2-dependent manner. Oncogene, 41(11), 1622-1633.
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7

Epigenetic Regulation of Osteoclastogenesis

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α‐Minimum Essential Medium (α‐MEM), Dulbecco's Modified Eagle Medium (DMEM), and protease inhibitor cocktail were purchased from Nacalai Tesque (Kyoto, Japan). Fetal bovine serum (FBS), penicillin‐streptomycin, and radioimmunoprecipitation assay (RIPA) buffer were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Human M‐CSF was purchased from PeproTech (Cranbury, NJ, USA). Bacteria‐derived soluble RANKL was purchased from Fujifilm Wako Pure Chemical (Osaka, Japan). A tartrate‐resistant acid phosphatase (TRAP) staining kit was purchased from Cosmo Bio (Tokyo, Japan). Polyinosinic–polycytidylic acid (pIpC) and polybrene transfection reagent were purchased from Sigma Aldrich (St. Louis, MO, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). Cell Counting Kit‐8 (CCK‐8) and Cell Cycle Assay Solution Blue were purchased from Doujindo (Kumamoto, Japan). The anti‐H3K4me3 antibody (polyclonal antibody, rabbit, 39159, RRID:AB_2615077) was purchased from Active Motif (Carlsbad, CA, USA). Anti‐H3K27me3 (polyclonal antibody, rabbit, 07‐449, RRID:AB_310624) and anti‐H3K27ac antibodies (monoclonal antibody, mouse, 05‐1334, RRID:AB_1977244) were from Millipore (Billerica, MA, USA).
Watanabe H., Okada H., Hirose J., Omata Y., Matsumoto T., Matsumoto M., Nakamura M., Saito T., Miyamoto T, & Tanaka S. (2022). Transcription Factor Hematopoietically Expressed Homeobox Protein (Hhex) Negatively Regulates Osteoclast Differentiation by Controlling Cyclin‐Dependent Kinase Inhibitors. JBMR Plus, 6(4), e10608.
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8

Chromatin Immunoprecipitation Profiling

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ChIP was performed using the ChIP-IT Express Enzymatic Magnetic Chromatin Immunoprecipitation Kit & Enzymatic Shearing Kit (Active Motif) according to the manufacturer's instructions. Macrophages or human lung fibroblasts were treated with vehicle (DMSO) or scriptaid (3 μg/ml) for 3 h and then fixed with 1% formaldehyde for 10 min. Chromatin was then prepared by enzymatic shearing and optimized according to the manufacturer's instructions. ChIP was performed using ChIP-IT Express on sheared chromatin from approximately 7.5 × 105 cells using a negative control IgG, an anti-RNA pol II antibody, an anti-H3K4me3 antibody, an anti-H3K9ac antibody, and an anti-p300 antibody (Active Motif). Real-time PCR was performed on DNA isolated from each of the ChIP reactions using specific primer pairs for the mouse Nox2 promoter regions located −75 to −1 upstream of Nox2 transcriptional start site (TSS), human Nox4 promoter regions located +185 to +344 downstream of Nox4 transcriptional start site (TSS), and the Nox5 promoter regions located −307 to −452 upstream of Nox5 transcriptional start site (TSS). The delta Ct was calculated by the relative fold enrichment for each antibody used versus IgG negative control.
Chen F., Li X., Aquadro E., Haigh S., Zhou J., Stepp D.W., Weintraub N.L., Barman S.A, & Fulton D.J. (2016). Inhibition of histone deacetylase reduces transcription of NADPH oxidases and ROS production and ameliorates pulmonary arterial hypertension. Free radical biology & medicine, 99, 167-178.
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9

ChIP-seq of LGR5+ Hair Follicle Stem Cells

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LGR5+ HFSCs were purified as previously described from anagen-synchronized HFs of Ring1a/b+/+ mice and were extracted following the protocol previously described. A total of 2.5 million cells were used for ChIP-seq as previously described (9 (link)). Sonicated chromatin was incubated with 10 μg of rabbit anti-RING1B antibody (homemade), anti-H2AK119Ub1 (8240, Cell Signaling Technology), anti-RYBP (AB3637, Millipore), and anti-CBX8 (39 (link)) overnight, and the immunocomplexes recovered using protein A–conjugated magnetic beads (Dynabeads, Life Technologies). Purified chromatin was decross-linked overnight in 0.1 M NaHCO3 and 1% SDS. Decross-linked DNA was purified. ISC-specific H3K4me3 ChIP-seq profile was obtained as previously described (9 (link)) using 5 μg of anti-H3K4Me3 antibody (catalog no. 39159, Active Motif).
Pivetti S., Fernandez-Perez D., D’Ambrosio A., Barbieri C.M., Manganaro D., Rossi A., Barnabei L., Zanotti M., Scelfo A., Chiacchiera F, & Pasini D. (2019). Loss of PRC1 activity in different stem cell compartments activates a common transcriptional program with cell type–dependent outcomes. Science Advances, 5(5), eaav1594.
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