Revision 11

#5246Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, W-S, IP, IHC-Bond, IHC-P, IF-F, IF-IC, FC-FP, ChIP, ChIP-seq, C&R, C&T

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

98

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q15910

Entrez-Gene Id:

2146

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 5 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.

The CUT&Tag dilution was determined using CUT&Tag Assay Kit #77552.

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:300
IHC Leica Bond 1:50 - 1:200
Immunohistochemistry (Paraffin) 1:50
Immunofluorescence (Frozen) 1:100 - 1:400
Immunofluorescence (Immunocytochemistry) 1:100 - 1:400
Flow Cytometry (Fixed/Permeabilized) 1:100 - 1:400
Chromatin IP 1:100
Chromatin IP-seq 1:100
CUT&RUN 1:100
CUT&Tag 1:100

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

For a carrier free (BSA and azide free) version of this product see product #16098.

Specificity / Sensitivity

Ezh2 (D2C9) XP® Rabbit mAb detects endogenous levels of total Ezh2 protein. This antibody does not cross-react with Ezh1 protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg354 of human Ezh2 protein.

Background

The polycomb group (PcG) proteins are involved in maintaining the silenced state of several developmentally regulated genes and contribute to the maintenance of cell identity, cell cycle regulation, and oncogenesis (1,2). Enhancer of zeste homolog 2 (Ezh2), a member of this large protein family, contains four conserved regions including domain I, domain II, and a cysteine-rich amino acid stretch that precedes the carboxy-terminal SET domain (3). The SET domain has been linked with histone methyltransferase (HMTase) activity. Moreover, mammalian Ezh2 is a member of a histone deacetylase complex that functions in gene silencing, acting at the level of chromatin structure (4). Ezh2 complexes methylate histone H3 at Lys9 and 27 in vitro, which is thought to be involved in targeting transcriptional regulators to specific loci (5). Ezh2 is deregulated in various tumor types, and its role, both as a primary effector and as a mediator of tumorigenesis, has become a subject of increased interest (6).

  1. Sellers, W.R. and Loda, M. (2002) Cancer Cell 2, 349-50.
  2. Visser, H.P. et al. (2001) Br J Haematol 112, 950-8.
  3. Chen, H. et al. (1996) Genomics 38, 30-7.
  4. Tonini, T. et al. (2004) Oncogene 23, 4930-7.
  5. Müller, J. et al. (2002) Cell 111, 197-208.
  6. Kleer, C.G. et al. (2003) Proc Natl Acad Sci U S A 100, 11606-11.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation IHC-Bond: IHC Leica Bond IHC-P: Immunohistochemistry (Paraffin) IF-F: Immunofluorescence (Frozen) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN C&T: CUT&Tag

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

限制使用

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专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品, (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

Revision 11
#5246

Ezh2 (D2C9) XP® Rabbit mAb

Western Blotting Image 1: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 Ezh2 (D2C9) XP® Rabbit mAb 对 MCF7、Neuro-2a 和 COS-7 细胞系提取物进行蛋白质印迹分析。
Western Blotting Image 1: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 Ezh2 (D2C9) XP® Rabbit mAb #5246 对 MCF-7 细胞的裂解物 (0.1 mg/mL) 进行 Simple Western™ 分析。虚拟泳道式图像(左图)显示一抗稀释比例为 1:10 和 1:50 时的靶标条带(如图所示)。对应的电泳图(右图)为一抗稀释比例在 1:10(蓝线)和 1:50(绿线)时沿毛细血管内分子量的化学发光结果。在还原条件下,使用 66-440 kDa 分离模块在 ProteinSimple(BioTechne 品牌)的 Jess™ Simple Western 仪器上进行该实验。
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Immunohistochemistry Image 1: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
在 Leica Bond Rx 上使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的人结肠腺癌组织进行免疫组织化学分析。
Immunohistochemistry Image 2: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
在 Leica BOND Rx 上使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的人 B 细胞非霍奇金淋巴瘤进行免疫组织化学分析。
Immunohistochemistry Image 1: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的人乳腺癌进行免疫组织化学分析。
Immunohistochemistry Image 2: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的人宫颈癌进行免疫组织化学分析。
Immunohistochemistry Image 3: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的人淋巴瘤进行免疫组织化学分析。
Immunohistochemistry Image 4: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 Ezh2 (D2C9) XP® 兔单克隆抗体对石蜡包埋的 4T1 小鼠同基因肿瘤进行免疫组织化学分析。
Immunohistochemistry Image 5: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的 小鼠大脑进行免疫组织化学分析。
Immunohistochemistry Image 6: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的小鼠肝脏进行免疫组织化学分析。
Immunohistochemistry Image 7: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的小鼠睾丸进行免疫组织化学分析。
Immunofluorescence Image 1: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 Ezh2 (D2C9) XP® Rabbit mAb(绿色)对小鼠海马(左图)和小脑(右图)进行共聚焦免疫荧光分析。肌动蛋白微丝由 DyLight 554 Phalloidin #13054(红色)标记。蓝色伪彩 = DRAQ5® #4084(DNA 荧光染料)。
Immunofluorescence Image 1: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 Ezh2 (D2C9) XP® Rabbit mAb(绿色)和S6 Ribosomal Protein (54D2) Mouse mAb #2317(蓝色)对 HeLa 细胞进行共聚焦免疫荧光分析。使用 DY-554 phalloidin(红色)标记肌动蛋白纤丝。
Flow Cytometry Image 1: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 EZH2 (D2C9) XP® Rabbit mAb 对未经处理(左图)和经抗人 CD3(10ug/ml,涂层平板)和抗人 CD28 (5ug/ml) 在 37ºC 下处理(右图)337 天的人外周血单核细胞进行流式细胞分析,并与抗人 CD3 抗体联合染色。使用 Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor 488 Conjugate) #4412 作为二抗。
Chromatin Immunoprecipitation Image 1: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对 HeLa 细胞中提取的交联染色质,在加入 Ezh2 (D2C9) XP® Rabbit mAb 或 Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb 后,进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。已发现 EZH2 和 H3K27me3 在染色质中相互结合。该图显示了 EZH2 和 H3K27me3 在 MYT1 基因中的结合。欲知其他 ChIP-seq 情况,请下载产品说明书。
Chromatin Immunoprecipitation Image 2: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对 HeLa 细胞中提取的交联染色质,在加入 Ezh2 (D2C9) XP® Rabbit mAb 或 Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb 后,进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。已发现 EZH2 和 H3K27me3 在染色质中相互结合。结果图显示在染色体 20(上图)内 EZH2 和 H3K27me3 两者的结合,包括 MYT1 基因(下图)。
Chromatin Immunoprecipitation Image 3: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 对 NCCIT 细胞中提取的交联染色质,在加入 Ezh2 (D2C9) XP® Rabbit mAb 或 Normal Rabbit IgG #2729 后,进行染色质免疫沉淀。使用 SimpleChIP® Human HoxA1 Intron 1 Primers #7707、SimpleChIP® Human HoxA2 Promoter Primers #5517 和 SimpleChIP® Human α Satellite Repeat Primers #4486 通过实时 PCR 对富集 DNA 进行定量分析。将每份样品中免疫沉淀的 DNA 的量表现为相对于所输入染色质总量(等于 1)的信号。
CUT and RUN Image 1: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652,通过 NCCIT 细胞和 Ezh2 (D2C9) XP® Rabbit mAb 进行 CUT&RUN。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在 Ezh2 的已知靶标基因 HoxA1 处结合(参见包含 CUT&RUN-qPCR 数据的其他结果图)。
CUT and RUN Image 2: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652,通过 NCCIT 细胞和 Ezh2 (D2C9) XP® Rabbit mAb 进行 CUT&RUN。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在染色体 7(上图)内的结合,包括 HoxA 基因(下图),HoxA 基因是 EZH2 靶标基因的已知基因簇(参见包含 CUT&RUN-qPCR 数据的其他结果图)。
CUT and RUN Image 3: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652,NCCIT 细胞和 Ezh2 (D2C9) XP® Rabbit mAb 或 Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362 进行 CUT&RUN。使用 SimpleChIP® Human HoxA1 Intron 1 Primers #7707、SimpleChIP® Human HoxA2 Promoter Primers #5517 和 SimpleChIP® Human α Satellite Repeat Primers #4486 通过实时 PCR 对富集 DNA 进行定量分析。将每份样品中免疫沉淀的 DNA 的量表现为相对于所输入染色质总量(等于 1)的信号。
CUT and Tag Image 1: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
CUT&Tag was performed with NCCIT cells and Ezh2 (D2C9) XP® Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across HOXA1, a known target gene of Ezh2 (see our ChIP-qPCR figure).
CUT and Tag Image 2: Ezh2 (D2C9) XP® Rabbit mAb Expand Image
CUT&Tag was performed with NCCIT cells and Ezh2 (D2C9) XP® Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figures show binding across the HOXA gene cluster (upper), and the HOXD gene cluster (lower), known target genes of Ezh2 (see our ChIP-qPCR figure).