Anti h3k27me2

Manufactured by Merck Group

Sourced in United States

Anti-H3K27me2 is a laboratory reagent used to detect the presence of dimethylation at lysine 27 of histone H3 protein. It is a specific antibody that binds to this histone modification, which is often associated with gene silencing and chromatin compaction. This product can be used in various experimental techniques, such as Western blotting, immunoprecipitation, and chromatin immunoprecipitation, to study the epigenetic regulation of gene expression.

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Lab products found in correlation

Anti h3k27me3, by Merck Group (4 mentions) Anti h3k27me1, by Merck Group (3 mentions) Anti β actin, by Merck Group (2 mentions) Anti h3k4me3, by Merck Group (2 mentions) Anti h3k4me2, by Merck Group (2 mentions) Anti h3k4me1, by Merck Group (2 mentions) Anti h3k9me3, by Merck Group (2 mentions) Anti h3k9me2, by Merck Group (2 mentions) Anti h3k9me1, by Merck Group (2 mentions) Nitrocellulose membrane, by Bio-Rad (2 mentions) Ab58632, by Abcam (1 mentions) Anti ezh2, by Merck Group (1 mentions) Dab substrate kit, by Vector Laboratories (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Anti suz12, by Cell Signaling Technology (1 mentions) Anti histone 3, by Merck Group (1 mentions) Anti eed, by Merck Group (1 mentions) Anti p16, by Santa Cruz Biotechnology (1 mentions) Anti parp, by Cell Signaling Technology (1 mentions) Anti gapdh, by Merck Group (1 mentions)

5 protocols using anti h3k27me2

Immunohistochemical Analysis of Growth Plate

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Growth plate sections were baked at 65 °C for 1 h, deparaffinized in xylene, rehydrated through ethanol series (100, 100, 95 and 70%) and rinsed with PBS. For all antibodies except Igfbp5, antigen retrieval was performed using proteinase K (100 μg ml⁻¹ in PBS) for 30 min at room temperature. For Igfbp5, heat-induced epitope retrieval with an alkaline buffer (10 mM Tris, 1 mM EDTA, 0.05% Tween-20, pH 9.0) was used. Endogenous peroxidase activity was blocked by 3% H₂O₂. Staining was performed using anti-Ezh1 (abcam, ab13665, 1:100), anti-Ezh2 (Millipore, 07-689, 1:1,000), anti-H3K27me3 (Millipore, 07-449, 1:1,000), anti-H3K27me2 (Millipore, 07-452, 1:1,000), anti-collagen X (abcam, ab58632, 1:1,000), anti-Cdkn2a (abcam, ab54210. 1:1,000), anti-Cdkn2b (Origene, TA312926, 1:100), anti-Igfbp3 (LifeSpan BioSciences, LS-C407922, 1:200) and anti-Igfbp5 (Thermo Scientific, PA5-37369, 1:50) antibodies, with a VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA) followed by the DAB Substrate kit (Vector Laboratories) according to the manufacturer’s instructions. Sections were mounted without counterstaining or counter-stained with methyl green.

Lui J.C., Garrison P., Nguyen Q., Ad M., Keembiyehetty C., Chen W., Jee Y.H., Landman E., Nilsson O., Barnes K.M, & Baron J. (2016). EZH1 and EZH2 promote skeletal growth by repressing inhibitors of chondrocyte proliferation and hypertrophy. Nature Communications, 7, 13685.

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Comprehensive Protein Expression Analysis

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Lysates were prepared in RIPA buffer [16 (link)], and western blotting was performed as described [17 (link)]. The following antibodies were used for western blot analysis: anti-Ezh2 (BD Biosciences #612666), anti-Suz12 (Cell Signaling 3737S), anti-Eed (Millipore 05–1320), anti-H3K27me1 (Millipore 07–448), anti-H3K27me2 (Millipore 07–452), anti-H3K27me3 (Millipore 07–449), anti-Histone 3 (Millipore 07–690), anti-β-actin (Sigma A5441), anti-Gapdh (Sigma G8795), anti-ERα (Millipore 07–690), anti-Ezh1 (Millipore 07–690), anti-PARP (Cell Signaling #9542), anti-p16 (Santa Cruz M-156), and anti-p19 (Rockland Immunochemicals #200-501-891). Secondary antibodies included HRP-conjugated anti-rabbit and anti-mouse (Southern Biotech), both 1:10,000.

Michalak E.M., Milevskiy M.J., Joyce R.M., Dekkers J.F., Jamieson P.R., Pal B., Dawson C.A., Hu Y., Orkin S.H., Alexander W.S., Lindeman G.J., Smyth G.K, & Visvader J.E. (2018). Canonical PRC2 function is essential for mammary gland development and affects chromatin compaction in mammary organoids. PLoS Biology, 16(8), e2004986.

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Histone Modification and Signaling Pathway Analysis

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Cells were harvested and lysed in RIPA buffer with protease inhibitors and 2 μM PMSF. Protein extracts were boiled in SDS sample buffer for 5 min, loaded directly onto a 4–12% SDS gel, transferred onto nitrocellulose membranes (Bio-Rad), blocked with 5% milk, and incubated with corresponding primary and secondary antibodies using standard protocols. The following antibodies were used: anti-H3K4me1 (Cat # 07-436, 1:2000 dilution), anti-H3K4me2 (Cat # 07-030, 1:4000 dilution), anti-H3K4me3 (Cat # 07-473, 1:4000 dilution), anti-H3K9me1 (Cat # 07-450, 1:2000 dilution), anti-H3K9me2 (Cat # 07-441, 1:3000 dilution), anti-H3K9me3 (Cat # 07-442, 1:3000 dilution), anti-H3K27me1 (Cat # 07-448, 1:2000 dilution), anti-H3K27me2 (Cat # 07-452, 1:4000 dilution), and anti-H3K27me3 (Cat # 07-449, 1:4000 dilution) from Millipore; rabbit anti-Jmjd3 (Cat # ab1022a, 1:500 dilution) from Abgent; and mouse anti-FLAG (1:5000 dilution), anti-HRP-FLAG (1:5000 dilution), and anti-β-actin (1:5000 dilution) from Sigma; anti-Smad1 (Cat # 6944, 1:500 dilution), anti-Smad2 (Cat # 5339, 1:500 dilution) and anti-Smad3 (Cat # 9523, 1:500 dilution) from Cell Signaling; and anti-Ash2L (Cat # ab50699, 1:500 dilution) from Abcam.

Li Q., Zou J., Wang M., Ding X., Chepelev I., Zhou X., Zhao W., Wei G., Cui J., Zhao K., Wang H.Y, & Wang R.F. (2014). Critical role of histone demethylase Jmjd3 in the regulation of CD4+ T cell differentiation. Nature communications, 5, 5780.

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Histone Modification Analysis in Arabidopsis

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Ten-day-old seedlings were ground in liquid nitrogen and the powder was boiled for 5 min in protein sample buffer. The proteins were resolved on a 15% SDS/PAGE gel and transferred onto nitrocellulose membranes (Bio-Rad). Then the membranes probed with anti-H3K27me3 (Millipore 07-449); anti-H3K27me2 (Millipore 07-452); anti-H3K27me1 (Millipore 07-448); anti-H3K4me3 (Millipore 07-473); anti-H3K4me2 (Millipore 07-030); anti-H3K4me1 (Millipore 07-436); anti-H3K9me2 (Millipore 07-441); anti-H3K9me1 (Millipore 07-450); anti-H3K36me3 (Abcam ab9050); anti-H3K36me2 (Millipore 07-274); anti-H3K36me1 (Millipore 07-548); anti-H3 (Abcam ab1791) or anti-GFP (Roche 11814460001) in TBST (137 mM NaCl, 20 mM Tris·HCl pH 7.6, 0.1% Tween-20). After three washes with TBST, the signals were detected with Immobilon Western Chemiluminescent HRP Substrate (Millipore) for histone antibodies or Super Signal West Dura Extended Duration Substrate (Thermo Fisher Scientific) for GFP antibody.

Zheng S., Hu H., Ren H., Yang Z., Qiu Q., Qi W., Liu X., Chen X., Cui X., Li S., Zhou B., Sun D., Cao X, & Du J. (2019). The Arabidopsis H3K27me3 demethylase JUMONJI 13 is a temperature and photoperiod dependent flowering repressor. Nature Communications, 10, 1303.

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ChIP Assay for Histone Modifications

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As described previously [21 (link),51 (link)], ChIP assays for histone methylation and HP1a recruitment were performed using an EZ-Magna ChIP™ G chromatin immunoprecipitation Kit (Millipore, California, United States). Briefly, monoclonal 293 T cells for stable expression of copGFP in 10 cm dishes were transiently transfected with 15 μg of various suppressor vectors. Forty eight hours after transfection, cells were cross-linked with 1% formaldehyde (Sigma, Missouri, United States) and harvested for immunoprecipitation. Antibodies used in ChIP assays included anti-H4K4Me3, anti-H3K9Me3, anti-H3K27Me2, and anti-HP1a (Millipore, California, United States). An aliquot of cell lysates was saved to serve as the input DNA control. After the reversal of crosslinking at 62°C for 2 hours and 95°C for 10 minutes, ChIP samples were purified and subjected to real-time qPCR. Individual ChIP assays were repeated three times to confirm the reproducibility of the qPCR. Real-time qPCR was performed using 2xKapa mixed with SYBR (Applied Biosystems, California, United States) on an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, California, United States) with pCMV primers (forward, 5’-gcggttttggcagtacatca-3’; reverse, 5’-gggcggagttgttacgacat-3’). Each sample was analyzed in quadruplicate.

Ma A.N., Wang H., Guo R., Wang Y.X., Li W., Cui J., Wang G., Hoffman A.R, & Hu J.F. (2014). Targeted gene suppression by inducing de novo DNA methylation in the gene promoter. Epigenetics & Chromatin, 7, 20.

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