Revision 4

#72335Store 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, IP, FC-FP, ChIP, C&R

REACTIVITY:

H Mk

SENSITIVITY:

Endogenous

MW (kDa):

96

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q9UBC3

Entrez-Gene Id:

1789

Product Information

Product Usage Information

For optimal ChIP 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.
Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100
Flow Cytometry (Fixed/Permeabilized) 1:400
Chromatin IP 1:100
CUT&RUN 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.

Specificity / Sensitivity

DNMT3B (E2Q3Z) Rabbit mAb recognizes endogenous levels of total DNMT3B protein. This antibody does not cross react with DNMT3A or DNMT1.

Species Reactivity:

Human, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu563 of human DNMT3B protein.

Background

Methylation of DNA at cytosine residues in mammalian cells is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting and development (1,2). Three families of mammalian DNA methyltransferases have been identified: DNMT1, DNMT2, and DNMT3 (1,2). DNMT1 is constitutively expressed in proliferating cells and functions as a maintenance methyltransferase, transferring proper methylation patterns to newly synthesized DNA during replication. DNMT3A and DNMT3B are strongly expressed in embryonic stem cells with reduced expression in adult somatic tissues. DNMT3A and DNMT3B function as de novo methyltransferases that methylate previously unmethylated regions of DNA. DNMT2 is expressed at low levels in adult somatic tissues and its inactivation affects neither de novo nor maintenance DNA methylation. DNMT1, DNMT3A, and DNMT3B together form a protein complex that interacts with histone deacetylases (HDAC1, HDAC2, Sin3A), transcriptional repressor proteins (RB, TAZ-1), and heterochromatin proteins (HP1, SUV39H1) to maintain proper levels of DNA methylation and facilitate gene silencing (3-8). Improper DNA methylation contributes to diseased states such as cancer (1,2). Hypermethylation of promoter CpG islands within tumor suppressor genes correlates with gene silencing and the development of cancer. In addition, hypomethylation of bulk genomic DNA correlates with and may contribute to the onset of cancer. DNMT1, DNMT3A, and DNMT3B are overexpressed in many cancers, including acute and chronic myelogenous leukemias, in addition to colon, breast, and stomach carcinomas (9-12).

  1. Hermann, A. et al. (2004) Cell. Mol. Life Sci. 61, 2571-87.
  2. Turek-Plewa, J. and Jagodziński, P.P. (2005) Cell. Mol. Biol. Lett. 10, 631-47.
  3. Kim, G.D. et al. (2002) EMBO J. 21, 4183-95.
  4. Fuks, F. et al. (2001) EMBO J. 20, 2536-44.
  5. Geiman, T.M. et al. (2004) Biochem. Biophys. Res. Commun. 318, 544-55.
  6. Rountree, M.R. et al. (2000) Nat. Genet. 25, 269-77.
  7. Pradhan, S. and Kim, G.D. (2002) EMBO J. 21, 779-88.
  8. Fuks, F. et al. (2003) Nucleic Acids Res. 31, 2305-12.
  9. Mizuno, S. et al. (2001) Blood 97, 1172-9.
  10. Robertson, K.D. et al. (1999) Nucleic Acids Res. 27, 2291-8.
  11. Xie, S. et al. (1999) Gene 236, 87-95.
  12. Kanai, Y. et al. (2001) Int. J. Cancer 91, 205-12.

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 BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP C&R: CUT&RUN

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.
SimpleChIP is a registered 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

除非 CST 的合法授书代表以书面形式书行明确同意，否书以下条款适用于 CST、其关书方或分书商提供的书品。 任何书充本条款或与本条款不同的客书条款和条件，除非书 CST 的合法授书代表以书面形式书独接受， 否书均被拒书，并且无效。

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

Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
仅供研究使用。不得用于诊断流程。
Revision 4
#72335

DNMT3B (E2Q3Z) Rabbit mAb

Western Blotting Image 1: DNMT3B (E2Q3Z) Rabbit mAb Expand Image
使用 DNMT3B (E2Q3Z) 兔单克隆抗体对 K-562、Huh7 和 COS-7 细胞系的提取物进行蛋白印迹分析。
Immunoprecipitation Image 1: DNMT3B (E2Q3Z) Rabbit mAb Expand Image
对 NCCIT 细胞提取物 DNMT3B 进行免疫沉淀。泳道 1 为 10% 输入，泳道 2 为兔 (DA1E) 单克隆抗体 IgG XP® 同型对照 #3900，泳道 3 为 DNMT3B (E2Q3Z) 兔单克隆抗体。使用 DNMT3B (E2Q3Z) 兔单克隆抗体和小鼠抗兔 IgG (特定构象) (L27A9) 单克隆抗体 (HRP 偶联物) #5127 进行蛋白印迹分析。
Flow Cytometry Image 1: DNMT3B (E2Q3Z) Rabbit mAb Expand Image
使用 DNMT3A (E2Q3Z) Rabbit mAb（实线）或浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900（虚线）对 NCCIT 细胞（绿色）、HCT116 WT 细胞（红色）或 HCT116 DNMT3B KO 细胞（蓝色）进行流式细胞分析。Anti-rabbit IgG (H+L)、F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 作为二抗。
Chromatin Immunoprecipitation Image 1: DNMT3B (E2Q3Z) Rabbit mAb Expand Image
使用 SimpleChIP® 酶解染色质免疫共沉淀试剂盒（磁珠法） #9005 对 NCCIT 细胞的交联染色质与 DNMT3B (E2Q3Z) 兔单克隆抗体#72335 或 正常兔 IgG #2729 进行染色质免疫沉淀。使用 SimpleChIP® 人源 EIF4G1 内含子 8 引物 #29118、人源 CROCC 内含子 1 引物 和 SimpleChIP® 人源 GAPDH 启动子引物 #4471 进行实时 PCR 来对富集的 DNA 进行定量。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: DNMT3B (E2Q3Z) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对 NCCIT 细胞和 DNMT3B (E2Q3Z) Rabbit mAb 或 DNMT3B (E8A8A) XP® Rabbit mAb #57868 进行 CUT&RUN 检测。使用 DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。图形显示在 HUWE1 内结合，HUWE1 是一种已知的 DNMT3B 靶标基因（参见包含 CUT&RUN-qPCR 数据的其他结果图）。
CUT and RUN Image 2: DNMT3B (E2Q3Z) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对 NCCIT 细胞和 DNMT3B (E2Q3Z) Rabbit mAb 或 DNMT3B (E8A8A) XP® Rabbit mAb #57868 进行 CUT&RUN 检测。使用 DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。该图显示在 DNMT3B 的已知靶基因 HUWE1（上图）和 SMARCA1（下图）内的结合（参见包含 CUT&RUN-qPCR 数据的其他结果图）。
CUT and RUN Image 3: DNMT3B (E2Q3Z) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对 NCCIT 细胞和 DNMT3B (E2Q3Z) Rabbit mAb 或 Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362 进行 CUT&RUN 检测。使用 human HUWE1 intron 26 primers、human ARHGEF2 promoter primers 和 SimpleChIP® Human GAPDH Exon 1 Primers #5516，通过实时 PCR 对富集的 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。
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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
仅供研究使用。不得用于诊断流程。