Revision 6

#5032Store 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, IHC-P, IF-IC, FC-FP, ChIP

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

200

Source/Isotype:

Rabbit IgG

UniProt ID:

#P26358

Entrez-Gene Id:

1786

Product Information

Product Usage Information

For optimal ChIP results, use 10 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.
Application Dilution
Western Blotting 1:1000
Immunohistochemistry (Paraffin) 1:50 - 1:200
Immunofluorescence (Immunocytochemistry) 1:100
Flow Cytometry (Fixed/Permeabilized) 1:50
Chromatin IP 1:50

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 #28754.

Specificity / Sensitivity

DNMT1 (D63A6) XP® Rabbit mAb detects endogenous levels of total DNMT1 protein. Species reactivity for IHC-P is human only.

Species Reactivity:

Human, Mouse, Rat, Monkey

Species predicted to react based on 100% sequence homology

Hamster, Bovine, Dog, Horse, Guinea Pig

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu985 of human DNMT1 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 IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP

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

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

Revision 6
#5032

DNMT1 (D63A6) XP® Rabbit mAb

Western Blotting Image 1: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
使用 DNMT1 (D63A6) XP® Rabbit mAb ,对 293 细胞和 F9 细胞提取物进行蛋白质印迹分析。
Immunohistochemistry Image 1: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
使用 DNMT1 (D63A6) XP® Rabbit mAb 对石蜡包埋的人卵巢浆液性腺癌组织进行免疫组织化学分析。
Immunohistochemistry Image 2: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
使用 DNMT1 (D63A6) XP® Rabbit mAb 对石蜡包埋的人软组织鳞状细胞癌组织进行免疫组织化学分析。
Immunohistochemistry Image 3: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
使用 DNMT1 (D63A6) XP® Rabbit mAb 对石蜡包埋的正常人胸腺进行免疫组织化学分析。
Immunohistochemistry Image 4: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
使用 DNMT1 (D63A6) XP® Rabbit mAb 对石蜡包埋的正常人睾丸进行免疫组织化学分析。
Immunohistochemistry Image 5: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
使用 DNMT1 (D63A6) XP® Rabbit mAb 对石蜡包埋的正常人淋巴结进行免疫组织化学分析。
Immunohistochemistry Image 6: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
使用 DNMT1 (D63A6) XP® Rabbit mAb(左图)或 DNMT1 Rabbit mAb(右图)对石蜡包埋的正常人胎盘进行免疫组织化学分析。这两种抗体可检测人 DNMT1 上独特的、不重叠的表位。使用两种抗体获得的相似染色模式有助于确认染色特异性。
Immunohistochemistry Image 7: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
以浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(右图)作为对照,使用 DNMT1 (D63A6) XP® Rabbit mAb(左图)对石蜡包埋的人乳腺导管癌细胞进行免疫组织化学分析。
Immunohistochemistry Image 8: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
使用 DNMT1 (D63A6) XP® Rabbit mAb 对未经处理(左图)或已经过 Doxycycline(10 µg/ml,72 小时;右图)处理的石蜡包埋的 HCT 116 细胞沉淀物进行免疫组织化学分析。
Immunofluorescence Image 1: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
使用 DNMT1 (D63A6) XP® Rabbit mAb(绿色)对 COS 细胞进行共聚焦免疫荧光分析。肌动蛋白纤丝用 DY-554 Phalloidin 进行标记(红色)。
Flow Cytometry Image 1: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
以浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线)作为对照,使用 DNMT1 (D63A6) XP® Rabbit mAb(实线)对 COS7 细胞进行流式细胞分析。Anti-rabbit IgG (H+L) F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 用作二抗。
Chromatin Immunoprecipitation Image 1: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,以来自均经多柔比星(1 μM,24 小时)处理的 HCT116 野生型细胞(左图)与 HCT116 DNMT3B/DNMT1 双敲除型细胞(右图)中的交联染色质和 DNMT1 (D63A6) XP® Rabbit mAb #5032 或 Normal Rabbit IgG #2729 进行染色质免疫沉淀。使用人 POMC 启动子引物和人 Stat2 启动子引物,通过实时 PCR 对富集的 DNA 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。