Anti h3k36me3

Manufactured by Active Motif

Anti-H3K36me3 is a chromatin immunoprecipitation (ChIP) grade antibody that recognizes the trimethylation of lysine 36 on histone H3. This post-translational modification is associated with actively transcribed regions of the genome.

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Lab products found in correlation

Anti h3k4me3, by Active Motif (3 mentions) Anti h3k36me2, by Cell Signaling Technology (2 mentions) Mabe191, by Abcam (2 mentions) Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibodies, by GE Healthcare (2 mentions) Anti his, by ZSGB-BIO (2 mentions) Anti β tubulin, by Cell Signaling Technology (2 mentions) Anti vinculin, by Cell Signaling Technology (2 mentions) Anti lamin b1, by Cell Signaling Technology (2 mentions) Anti β actin, by Abcam (2 mentions) Anti ha, by BioLegend (2 mentions) Anti h3k36me2, by Active Motif (2 mentions) Anti h3, by Abcam (2 mentions) Chip it express kit, by Active Motif (1 mentions) Anti acetyl histone h3, by Merck Group (1 mentions) Rabbit igg, by Santa Cruz Biotechnology (1 mentions) Sodium pyruvate, by Corning (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Anti h4k12ac, by Abcam (1 mentions) Anti brdu, by BD (1 mentions) Anti h3k4me3, by Abcam (1 mentions)

8 protocols using anti h3k36me3

Western Blot Analysis of Histone Modifications

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Fractionated or whole cell lysates were resolved by SDS-PAGE, transferred to a nitrocellulose or PVDF membrane, blocked in 5% non-fat milk in PBS plus 0.5% Tween-20, probed with primary antibodies, and detected with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (GE Healthcare). Primary antibodies were: anti-H3K36me2 (Cell Signaling Tech, 2901), anti-H3K36me3 (Active Motif, 61101), anti-NSD1 (Abbexa, abx135901), anti-NSD2 (Millipore Sigma, MABE191), anti-SETD2 (Abcam, ab31358), anti-DNMT3A (Abcam, ab188470), anti-Vinculin (Cell Signaling Tech, 13901), anti-His (ZSGB-Bio,TA-02), anti-Lamin B1 (Cell Signaling Tech, 12586), anti-β-Tubulin (Cell Signaling Tech, 2128), anti-β-actin (Abcam, ab8224), anti-H3 (Abcam, ab1791) and anti-HA (Biolegend, 901501). The specificities of anti-H3K36me3 and anti-H3K36me2 antibodies were validated using Setd2 knockout and Nsd1/2 double knockout cell lines.

Weinberg D.N., Papillon-Cavanagh S., Chen H., Yue Y., Chen X., Rajagopalan K.N., Horth C., McGuire J.T., Xu X., Nikbakht H., Lemiesz A.E., Marchione D.M., Marunde M.R., Meiners M., Cheek M., Keogh M.C., Bareke E., Djedid A., Harutyunyan A.S., Jabado N., Garcia B.A., Li H., Allis C.D., Majewski J, & Lu C. (2019). H3K36me2 recruits DNMT3A and shapes the intergenic DNA methylation landscape. Nature, 573(7773), 281-286.

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ChIP-IT Express Protocol for Epigenetic Profiling

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The ChIP-IT Express kit (Active Motif) was used for ChIP assay in this study following the manufacturer's protocol. The additional antibodies used in ChIP assay were rabbit IgG (Santa Cruz Biotechnology, sc-2027), anti-acetyl-histone H3 (Millipore, 06-599), anti-H3K36me2 (Active Motif, 39255), anti-H3K4me3 (Active Motif, 39159), and anti-H3K36me3 (Active Motif, 61101). These antibodies are all ChIP grade and have been validated by their companies. The ChIP PCR primers for amplification of ERα/ESR1 gene next to the promoter region (A) are: Forward, 5′-CCCACTCAACAGCGTGTCT-3′ Reverse, 5′-CTGCAGGAAAGGCGACAG-3′. The ChIP primers for amplification of ERα gene in the coding region (B) are: Forward, 5′-GAAGAAGCATGGGTAAATGTCA-3′ Reverse, 5′-TCAGCCCTGAACCCAGTG-3′.

Feng Q., Zhang Z., Shea M.J., Creighton C.J., Coarfa C., Hilsenbeck S.G., Lanz R., He B., Wang L., Fu X., Nardone A., Song Y., Bradner J., Mitsiades N., Mitsiades C.S., Osborne C.K., Schiff R, & O'Malley B.W. (2014). An epigenomic approach to therapy for tamoxifen-resistant breast cancer. Cell Research, 24(7), 809-819.

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Mouse E14 ES Cell Culture and Histone Antibodies

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Mouse E14 ES cells (a gift from T. Fazzio, University of Massachusetts Medical School, Worcester, MA) were cultured in Dulbecco’s modified Eagle’s medium (Corning) supplemented with 15% (v/v) fetal bovine serum, 1% penicillin/streptomycin (Invitrogen), 1 mM sodium pyruvate (Cellgro), 2 mM l-glutamine (Cellgro), 1% MEM nonessential amino acids (Invitrogen), 55 μM β-mercaptoethanol (Sigma-Aldrich), and mouse leukemia inhibitory factor (10 ng/ml) on gelatin-coated dishes in a 37°C incubator with a humidified, 5% CO₂ atmosphere. The antibodies used in this study include anti-H4K20me2 (Diagenode, C15200205), anti-H4K12ac (Abcam, ab46983), anti-H3K36me3 (Active Motif, 61021), anti-H3K4me3 (Abcam, ab8580), and anti-BrdU (BD Biosciences, 555627). Other antibodies including anti-H3K56ac, anti-H3, anti-H4, anti-H2A, and anti-H2B were generated by our lab using a synthetic peptide or full-length recombinant proteins.

Li Z., Hua X., Serra-Cardona A., Xu X., Gan S., Zhou H., Yang W.S., Chen C.L., Xu R.M, & Zhang Z. (2020). DNA polymerase α interacts with H3-H4 and facilitates the transfer of parental histones to lagging strands. Science Advances, 6(35), eabb5820.

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Western Blot Analysis of Histone Modifications

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Cells were harvested and lysed in ice-cold radio immunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitor cocktail and phosphatase inhibitors (Sigma, MO, USA). Protein concentrations were determined by BCA kit (Bio-Rad, Hercules, CA, USA). Briefly, 10 μg of protein were loaded on 12% tris-glycine polyacrylamide gel prior to being transferred to a PVDF membrane on ice. After transfer, membranes were blocked with 5% milk in tris-buffered saline with 0.1% Tween-20 (TBST) for 30 min. Membranes were then incubated with primary antibodies: anti-H3K36me3 (1:1000, Active Motif, Carlsbad, CA), anti-CS (1:1000, Cell Signaling, Danvers, MS), and anti-PGC1α (1:1000, Cell Signaling) at 4 °C overnight. After washing with TBST, membranes were incubated with goat anti-rabbit/mouse HRP (1:10,000, Jackson Immunoresearch, West Grove, PA) for 1 h at room temperature. After washing with TBST, membranes were observed by chemiluminescent kit (Thermo Scientific, Carlsbad, CA, USA).

Liu J., Hanavan P.D., Kras K., Ruiz Y.W., Castle E.P., Lake D.F., Chen X., O’Brien D., Luo H., Robertson K.D., Gu H, & Ho T.H. (2018). Loss of SETD2 induces a metabolic switch in renal cell carcinoma cell lines toward enhanced oxidative phosphorylation. Journal of proteome research, 18(1), 331-340.

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Chromatin Immunoprecipitation Profiling Antibodies

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The following commercially available antibodies were used:
Anti-H3 (1:5.000, #61475), Anti-H3K36me3 (1:4.000, #61102), Anti-H3K36me2 (1:1.000, #39255), Anti-H3K36me1 (1:1.000, #61351), Anti-H3K4me3 (1:500, #61379) and anti-H2A (1:1.000, #39209) were from Active Motif. Anti-H3K27me3 (1:1.000, C15410069), Anti-H3K79me3 (1:1.000, C15310068), anti-H2A.Zac (2 µg/IP, C15410202-050) and Anti-H3K9me3 (1:1.000, C15410056) were from Diagenode. Anti-H4K20me3 (1:1.000, ab9053), Anti-H3 (1:30.000, ab1791), anti-MTA1 (1:1.000, ab71153 or 3 µg/IP, ab50209), anti-CHD4 (1:1.000 or 2 µg/IP, ab70469), anti-HDAC2 (1:5.000, ab124974) anti-RBBP4 (1:1.000, ab488), anti-RBBP7 (1:1.000, ab3535), Anti-H3K27ac (2 µg/IP, ab4729), anti-H2A.Z (1:3.000, ab4174), and anti-MBD3 (1:5.000, ab157464) were from Abcam. Anti-PWWP2A (1:1.000, NBP2-13833) from Novus (Acris). Anti-HA-HRP (1:40.000, 2999S) and anti-HDAC1 (1:20.000, 5356S) from Cell Signaling Technology. Anti-FLAG M2 (1:80.000 or 3 µg/IP, F1804) and anti-mouse IgG (3 µg/IP, I8765) from Sigma-Aldrich.
The following secondary antibodies were used:
Anti-rabbit IRDye 800CW (1:10.000, 926-32211) and anti-mouse IRDye 680RD (1:10.000 926‐68070) from LI-COR Biosciences. Anti-mouse-HRP (1:10.000, M114) from Leinco Technologies. Anti-mouse and anti-rabbit HRP-linked antibodies (1:10.000, NA931 and NA934) from VWR.

Link S., Spitzer R.M., Sana M., Torrado M., Völker-Albert M.C., Keilhauer E.C., Burgold T., Pünzeler S., Low J.K., Lindström I., Nist A., Regnard C., Stiewe T., Hendrich B., Imhof A., Mann M., Mackay J.P., Bartkuhn M, & Hake S.B. (2018). PWWP2A binds distinct chromatin moieties and interacts with an MTA1-specific core NuRD complex. Nature Communications, 9, 4300.

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ChIP-qPCR analysis of histone marks

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ChIP experiments were performed as previously described with minor modifications (41 (link)). Briefly, total chromatin was extracted from 10 DAG seedlings and immuno-precipitated using anti-HA (Santa Cruz Biotechnology, #sc-7392) and normal mouse IgG as a control (Santa Cruz Biotechnology, #sc-2025), or anti-H3Ace3 (Millipore, #06-599), anti-H4K5Ace (Active Motif #39699), anti-H3K4me3 (Active Motif #61379) and anti-H3K36me3 (Active Motif #61021), with anti-H3 (Abcam, #ab1791) as a control. DNA fragments were recovered by phenol-chloroform extraction and ethanol precipitation. Quantitative PCR with locus-specific primers (Supplementary Table S1) was performed to measure the amounts of FT relative to that of the constitutively expressed ACTIN2 (AT3G18780) on ABI PRISM 7900HT sequence detection system (Applied Biosystems) using KAPA SYBR FAST ABI Prism qPCR Master Mix (KAPA Biosystems).

Xu Y., Gan E.S., Zhou J., Wee W.Y., Zhang X, & Ito T. (2014). Arabidopsis MRG domain proteins bridge two histone modifications to elevate expression of flowering genes. Nucleic Acids Research, 42(17), 10960-10974.

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Histone Modification Profiling Protocol

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Rabbit polyclonal anti-Histone H3 (Abcam, Cat. #ab1791); Rabbit polyclonal anti-Histone H3K4me3 (Abcam, Cat. #ab8580); Rabbit monoclonal anti-Histone H3K27me3 (Cell Signaling Technology, Cat. #9733); Rabbit polyclonal anti-Histone H3K27ac (Abcam, Cat. #ab4729); Rabbit monoclonal anti-H3K36me2 (Cell Signaling Technology, Cat. #2901); Rabbit polyclonal anti-H3K36me3 (Active motif, Cat. #61101); Rabbit polyclonal anti-Histone H3K4me1 (Abcam, Cat. #ab8895); Rabbit monoclonal anti-HDAC1 (Cell Signaling Technology, Cat. #34589); Mouse monoclonal anti-β-Tubulin (Cell Signaling Technology, Cat. #86298); Rabbit polyclonal anti-Brd4 (Abcam, Cat. #ab75898); Mouse monoclonal anti-FLAG (GenScript, Cat. #A00187); Rabbit polyclonal anti-NSD1(Abbexa, Cat. #135901).

Fang Y., Tang Y., Zhang Y., Pan Y., Jia J., Sun Z., Zeng W., Chen J., Yuan Y, & Fang D. (2021). The H3K36me2 methyltransferase NSD1 modulates H3K27ac at active enhancers to safeguard gene expression. Nucleic Acids Research, 49(11), 6281-6295.

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Western Blot Analysis of Histone Modifications

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