Ab85392

Manufactured by Abcam

Ab85392 is a laboratory equipment product offered by Abcam. It serves a core functional purpose, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

Automatically generated - may contain errors

Lab products found in correlation

Ab9110, by Abcam (2 mentions) Ab1791, by Abcam (2 mentions) Ab8580, by Merck Group (1 mentions) H6908, by Abcam (1 mentions) Viia7 instrument, by Thermo Fisher Scientific (1 mentions) Ab172730, by Abcam (1 mentions) Anti c ebpβ sc 150, by Santa Cruz Biotechnology (1 mentions) Protein g coated magnetic dynabeads, by Thermo Fisher Scientific (1 mentions) F7425, by Merck Group (1 mentions) Ab6002, by Abcam (1 mentions) Chromaflash chromatin extraction kit, by Epigentek (1 mentions) Lightcycler faststart dna master sybr green 1, by Roche (1 mentions) Lightcycler 96 instrument, by Roche (1 mentions) Cross linking chip protocol, by Abcam (1 mentions) Qiaquick pcr purification kit, by Qiagen (1 mentions) Nitrocellulose membrane, by Cytiva (1 mentions) A5316, by Merck Group (1 mentions) Anti h3k27me1, by Merck Group (1 mentions) Ab8580, by Abcam (1 mentions) Ab4729, by Abcam (1 mentions)

7 protocols using ab85392

ChIP-Seq Profiling of Histone Marks

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Standard ChIP assays were performed as described in Supplemental Experimental Procedures with antibodies against H3K27me3 (Millipore, #07-449), H3K4me3 (Abcam, #ab8580), HA tag (Sigma, #H6908), Jmjd3 (Abcam, #ab85392) or normal rabbit IgG.

Pan D., Huang L., Zhu L.J., Zou T., Ou J., Zhou W, & Wang Y.X. (2015). Jmjd3-mediated H3K27me3 dynamics orchestrate brown fat development and regulate white fat plasticity. Developmental cell, 35(5), 568-583.

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Molecular Profiling of Stem Cell Differentiation

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qRT-PCR was performed using Taqman “Assays on Demand” (Invitrogen) on a Viia7 instrument (Life Technologies). All assays were performed in triplicate, normalized to GAPDH, and analyzed using the ΔΔCT method. Data are representative of multiple independent experiments. Immunoblotting, immunoprecipitations, and immunostaining were performed as previously described (Singh et al., 2012 (link)), with antibodies raised against MLL1 (A300-086A), MLL2 (A300-113A, Bethyl Laboratories); MENIN (ab2605), JMJD3 (ab85392, Abcam), WDR82 (kind gift from David Skalnik, IUPUI School of Science); pSMAD2 (3104S), pSerine-CDKs Substrate, P-S-100 (9477), pThreonine-Proline (9391) (Cell Signaling Technology); OCT4 (sc-8628), CDK2 (sc-163) (Santa Cruz Biotechnology); BRACHYURY (AF2085), SOX17 (AF1924) (R&D systems); FOXA2 (07-633, Millipore).

Singh A.M., Sun Y., Li L., Zhang W., Wu T., Zhao S., Qin Z, & Dalton S. (2015). Cell-Cycle Control of Bivalent Epigenetic Domains Regulates the Exit from Pluripotency. Stem Cell Reports, 5(3), 323-336.

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Protein Immunoprecipitation and Western Blot

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The cells were lysed in 1 ml lysis-buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, 2 mM EDTA, 25 mM glycerophosphate, 100 mM NaF, 200 mM Na₃VO₄, 1 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mM PMSF). According to the manufacturer’s protocol, the insoluble material in cell lysates was removed via centrifugation, and the recovered supernatant was immunoprecipitated with Protein G-coated magnetic Dynabeads (Invitrogen) and 10 µg of each of the following antibodies separately: anti-JMJD3 (ab85392, Abcam); anti-C/EBPβ (sc-150×, Santa Cruz); anti-FLAG (F7425, Sigma), and control IgG (ab172730, Abcam). To be prepared for western blotting analysis, firstly the immunoprecipitates were washed four times with 1 ml lysis-buffer, then resuspended in 50 µl 2× Laemmli sample buffer (125 mM Tris pH 6.8, 4% SDS, 25% glycerol, 0.1% bromophenol blue, 5% β-mercaptoethanol), and finally boiled at 95 °C for 10 min. Control (INPUT) was made by 50 µl cell lysates processed for all steps except for the incubation with Protein G-coated magnetic Dynabeads and antibodies. The uncropped scans of blots are shown in the Supplementary Fig. 7.

Yu S.H., Zhu K.Y., Chen J., Liu X.Z., Xu P.F., Zhang W., Yan L., Guo H.Z, & Zhu J. (2018). JMJD3 facilitates C/EBPβ-centered transcriptional program to exert oncorepressor activity in AML. Nature Communications, 9, 3369.

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Chromatin Immunoprecipitation for Tfam Regulation

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A chromatin immunoprecipitation assay was performed as previously described 36 (link). Chromatin preparation was done from the osteoblasts using the ChromaFlash Chromatin Extraction Kit (Epigentek, Farmingdale, NY, USA), according to the manufacturer's instructions. Briefly, the prepared chromatin was immunoprecipitated with anti-KDM6b antibody-ChIP grade (ab85392, Abcam) or anti-Histone H3 (tri methyl K27) antibody- ChIP Grade (ab6002, Abcam) overnight. It was amplified by PCR reaction using LightCycler® FastStart DNA Master SYBR Green I (Roche Life Science) and PCR instrumentation LightCycler® 96 Instrument (Roche Life Science). Primers were designed for the Tfam promoter. The primer pairs used to amplify sequences surrounding predicted KDM6b or H3K27me3 binding sites at the Tfam locus are presented in Table S1.

Behera J., Ison J., Voor M.J, & Tyagi N. (2021). Probiotics Stimulate Bone Formation in Obese Mice via Histone Methylations. Theranostics, 11(17), 8605-8623.

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ChIP-seq Analysis of Histone Modifications

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ChIP assays were performed according to the standard cross-linking ChIP protocol (Abcam) with minor modifications. For ChIP using H3K27me3 antibody, 200 each wild-type embryos; control morphants; jmjd3 morphants and HA-jmjd3-overexpression embryos at 22 hpf were used. For ChIP using H3K4me3 antibody, 200 each control morphants and jmjd3 morphants at 22 hpf were used. For ChIP using HA antibody, 200 each wild-type embryos and HA-jmjd3-overexpression embryos at 22 hpf were used. Zebrafish embros were enzymatically dechorionated, and cells were harvested and crosslinked with 1% formaldehyde for 10 minutes at room temperature. After sonication, the soluble chromatins were incubated with 5 ug antibodies specific for H3K27me3 (ab85392 Abcam) or HA (ab9110, Abcam). Chromatin immunocomplexes were then precipitated with Protein A (Millipore, 16-661) or Protein G (Millipore, 16-662). The immunoprecipitated complex was washed, and DNA was extracted and purified by QIAquick PCR Purification Kit (Qiagen). ChIP DNA was analyzed by qPCR using specific primers against genomic regions of interest, and the data were normalized by input DNA. The results were derived from three independent experiments. The primer sequences are available in Table S2.

Yu S.H., Zhu K.Y., Zhang F., Wang J., Yuan H., Chen Y., Jin Y., Dong M., Wang L., Jia X.E., Gao L., Dong Z.W., Ren C.G., Chen L.T., Huang Q.H., Deng M., Zon L.I., Zhou Y., Zhu J., Xu P.F, & Liu T.X. (2018). The histone demethylase Jmjd3 regulates zebrafish myeloid development by promoting spi1 expression. Biochimica et biophysica acta, 1861(2), 106-116.

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Histone Modifications Western Blot Analysis

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Cells were washed with PBS and collected in Laemmli buffer directly. Protein samples were separated by 4-20% SDS-PAGE precast gel (Bio-Rad #456-1096) at 200 V for 30 min, and then transferred to nitrocellulose membranes (Whatman, Dassel, Germany) at 30V for 70 min. Membranes were blocked with 4% non-fat milk in PBS-0.1%Tween buffer (PBST) for 30min at room temperature. Primary antibodies anti-H3K27me3 (Millipore #07-449), anti-H3K27me1 (Millipore #07-448), anti-histone H3 (Abcam #ab1791), anti-JMJD3 (Abcam # ab85392), UTX (Abcam # ab36938), and anti-α-actin (Sigma #A5316) were diluted in 4% non-fat milk in PBST and incubated with membranes overnight at 4°C. Membranes were wash for 3 times with PBST and then incubated with secondary antibodies (Millipore) for 1 h at room temperature. Membranes were then washed with PBST 3 times and visualized using ECL reagent. Western blots analysis was performed using software Image J.

Morozov V.M., Li Y., Clowers M.M, & Ishov A.M. (2017). Inhibitor of H3K27 demethylase JMJD3/UTX GSK-J4 is a potential therapeutic option for castration resistant prostate cancer. Oncotarget, 8(37), 62131-62142.

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Chromatin Immunoprecipitation Profiling

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ChIP experiments were performed as described previously^{12 (link)}. Dissociated cells were crosslinked with PFA or double crosslinked with DSG (Pierce) and PFA, and sonicated to fragments (200–1,000 bp). Antibodies used were against H3K27me3 (07-449, Millipore), H3K4me3 (ab8580, Abcam), H3K27Ac (ab4729, Abcam), histone H3 (ab1791, Abcam), SUZ12 (#3737, Cell Signaling), EZH2 (612667, BD Bioscience), Jmjd3 (ab85392, Abcam)^{70 (link)}, WDR5 (A302-429A, Bethyl Laboratories), HA (ab9110, Abcam) and FLAG (F1804, M2, Sigma). Rabbit IgG was used as a negative control. Precipitated DNA was purified and subjected to real-time PCR.

Shi X., Zhang Z., Zhan X., Cao M., Satoh T., Akira S., Shpargel K., Magnuson T., Li Q., Wang R., Wang C., Ge K, & Wu J. (2014). An epigenetic switch induced by Shh signalling regulates gene activation during development and medulloblastoma growth. Nature communications, 5, 5425.

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