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Datasheet
Monoclonal Antibody to Dnmt1 (DNA methyltransferase 1)
Cat.No IMG-261A
Description Dnmt1
Format Purified
Unit 0.1 mg
Price(USD) 240.00
MSDS
Catalog No
:
IMG-261A
Contents
:
100 ug in 200 ul PBS containing 0.05% BSA and 0.05% sodium azide. Sodium azide is highly toxic.
Isotype
:
Mouse IgG1, Kappa
Clone
:
60B1220.1
Purification
:
Protein G Chromatography
Species
:
Human, Mouse, Zebrafish
Host
:
Mouse
Application
ChIP: please see product citation Yu-Wei Leu et al for details IHC (paraffin): 1-2 ug/ml IP: see product citations listing IP for details. Western blot analysis: 0.1-0.5 ug/ml Storage Store at 4ºC. For long-term storage, store at -20ºC. Recommended Positive Control : human kidney (IHC), mouse ES or NIH 3T3 cell lysate (WB)
Background Methylation of DNA at cytosine residues plays an important role in regulation of gene expression, genomic imprinting and is essential for mammalian development. Hypermethylation of CpG islands in tumor suppressor genes or hypomethylation of bulk genomic DNA may be linked with development of cancer. To date, 3 families of mammalian DNA methyltransferase genes have been identified which include Dnmt1, Dnmt2 and Dnmt3. Dnmt1 is constitutively expressed in proliferating cells and inactivation of this gene causes global demethylation of genomic DNA and embryonic lethality. Dnmt2 is expressed at low levels in adult tissues and its inactivation does not affect DNA methylation or maintenance of methylation. The Dnmt3 family members, Dnmt3a and Dnmt3b, are strongly expressed in ES cells but their expression is down regulated in differentiating ES cells and is low in adult somatic tissue. Dnmt1 co-purifies with the retinoblastoma (Rb) tumour suppressor gene product, E2F1, and HDAC1. Dnmt1 also cooperates with Rb to repress transcription from promoters containing E2F-binding sites suggesting a link between DNA methylation, histone deacetylase and sequence-specific DNA binding activity, as well as a growth-regulatory pathway that is disrupted in nearly all cancer cells.
IMG-261A recognizes Dnmt1, a 1616 amino acid protein according to GenBank no. NP_001370.
Antigen
This antibody was raised against a synthetic peptide corresponding to amino acids 637-650 (EKDDREDKENAFKR) of human Dnmt1 (Genbank Accession No. NP_001370). It will cross react with mouse Dnmt1.
Application Notes
This antibody can be used in immunohistochemistry at 1-2 ug/ml in human kidney cells.
For the western blot data pictured below, 0.5 ug/ml of IMG-261A was used for testing the human cell line and 0.1 ug/ml was used for testing the recombinant protein and the mouse cell lines.
Western blot analysis of Dnmt1 in 2102EP (human embryonic carcinoma) in the A) absence and B) presence of immunizing peptide, recombinant human Dnmt1 protein in the C) absence and D) presence of immunizing peptide, NIH 3T3 (mouse embryonic fribroblast) in the E) absence and F) presence of immunizing peptide, and D3 (mouse embryonic stem cell) in the G) absence and H) presence of immunizing peptide using IMG-261A. IMGENEX’s goat anti-mouse Ig HRP secondary antibody (IMGENEX, 20101) and PicoTect ECL substrate solution (IMGENEX, 10087K) were used for this test.
IHC analysis of Dnmt1 using IMG-261A at 2 ug/ml on formalin-fixed, paraffin-embedded medullar kidney tissue sections
Reference 1. Bestor. T., et al.. J.Mol. Biol. 203: 971-983 (1988). 2. Yen RW, Vertino PM, Nelkin BD et al. Nucl. Acids Res. 20: 2287-2291 (1992). 3. Xie, S. et al. Gene 236: 87-95 (1999). 4. Okano M, Bell DW, Haber DA,a nd Li E. Cell 99: 247-257 (1999). 5. Reik W. et al. J. Nat. Genet 23: 380-382 (1999). 6. Robertson KD, et al. Nat Genet 25(3):338-342 (2000).
Product Citations 1. Decrease of DNA methyltransferase 1 expression relative to cell proliferation in transitional cell carcinoma. Fumihiro Kimura, Hans-Helge Seifert, Andrea R. Florl, Simon Santourlidis, Christine Steinhoff , Sandra Swiatkowski , Csaba Mahotka , Claus-Dieter Gerharz , and Wolfgang A. Schultz. International J Cancer, 104 (5): 568-578 (2003). WB: human bladder transitional cell carcinoma cell lines and primary cultured normal human uroepithelial cells (Fig. 2). 2. Components of a Pathway Maintaining Histone Modification and Heterochromatin Protein 1 Binding at the Pericentric Heterochromatin in Mammalian Cells. Huawei Xin, Ho-Guen Yoon, Prim B. Singh, Jiemin Wong, and Jun Qin. J. Biol. Chem., Vol. 279, 10: 9539-9546 (2004). 3. RNA interference-mediated knockdown of DNA methyltransferase 1 leads to promoter demethylation and gene re-expression in human lung and breast cancer cells. M Suzuki, Sunaga N, Shames DS, Toyooka S, Gazdar AF and Minna JD. Cancer Research 64:3137-3143 (2004). WB (Fig 1): NCI-H1299 non small cell lung cancer and HCC1954 breast cancer human cell lines. Cells treated with DNMT1 siRNA showed a dramatic reduction of DNMT1 expression.
Note: The specificity of the DNMT1 (IMG-261A) was siRNA validated in Fig 1 by WB in the NCI-H1299 and HCC1954 cell lines.
4. Induction of DNA methylation and gene silencing by short interfering RNAs in human cells. Hiroaki Kawasaki, Kazunari Taira. Nature 431: 211-217 (2004). 5. Farnesyltransferase inhibitor (L-744,832) restores TGF-b type II receptor expression and enhances radiation sensitivity in K-ras mutant pancreatic cancer cell line MIA PaCa-2. Rachael A Alcock, Swatee Dey, Damodaran Chendil, Mohammed S Inayat, Mohammed Mohiuddin, George Hartman, Lee K Chatfield, Vincent S Gallicchio, Mansoor M Ahmed. Oncogene 21, 7883-7890 (2002). 6. The nucleolar remodeling complex NoRC mediates heterochromatin formation and silencing of ribosomal gene transcription. Raffaella Santoro, Junwei Li, Ingrid Grummt. Nature Genetics 32, 393-396 (2002). 7. The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases. Chi-Neu Tsai, Chia-Lung Tsai, Ka-Po Tse, Hwan-You Chang, and Yu-Sun Chang. PNAS, 99: 10084-10089 (2002). 8. Age-dependent DNA methylation changes in the ITGAL (CD11a) promoter . Zhiyong Zhang, Chun Deng, Qianjin Lu and Bruce Richardson. Mechanisms of Ageing and Development 123: 1257-1268 (2002). 9. Double RNA Interference of DNMT3b and DNMT1 Enhances DNA Demethylation and Gene Reactivation. Yu-Wei Leu, Farahnaz Rahmatpanah, Huidong Shi, Susan H. Wei, Joseph C. Liu, Pearlly S. Yan and Tim Hui-Ming Huang. Cancer Research 63: 6110-6115 (2003). 10. Analysis of mammalian proteins involved in chromatin modification reveals new metaphase centromeric proteins and distinct chromosomal distribution patterns. Jeffrey M. Craig, Elizabeth Earle, Paul Canham, Lee H. Wong, Melissa Anderson, and K.H. Andy Choo. Hum. Mol. Genet., 12: 3109-3121 (2003). 11. Lsh, a member of the SNF2 family, is required for genome-wide methylation. Kathleen Dennis, Tao Fan, Theresa Geiman, Qingsheng Yan, and Kathrin Muegge. Genes Dev. 15: 2940-2944 (2001). 12. Loss of Estrogen Receptor Signaling Triggers Epigenetic Silencing of Downstream Targets in Breast Cancer. Yu-Wei Leu, Pearlly S. Yan, Meiyun Fan, Victor X. Jin, Joseph C. Liu, Edward M. Curran, Wade V. Welshons, Susan H. Wei, Ramana V. Davuluri, Christoph Plass, Kenneth P. Nephew, and Tim H-M. Huang. Cancer Research 64, 8184-8192 (2004). (ChIP Assay) 13. Deregulation of DNA methyltransferases and loss of parental methylation at the insulin-like growth factor II (Igf2)/H19 loci in p53 knockout mice prior to tumor development. IY Park , Sohn BH, Choo JH, Joe CO, Seong JK, Lee YIK, JChung JH. J Cellular Biochem 94:585-596 (2005). IMGENEX antibodies cited for WB (Fig 1), mouse thymus and liver:
DMNT1 (IMG-261A)
DNMT3a (IMG-268A)
14. 5-Aza-Deoxycytidine Induces Selective Degradation of DNA Methyltransferase 1 by a Proteasomal Pathway That Requires the KEN Box, Bromo-Adjacent Homology Domain, and Nuclear Localization Signal. Kalpana Ghoshal, Jharna Datta, Sarmila Majumder, Shoumei Bai, Huban Kutay, Tasneem Motiwala, and Samson T. Jacob. Mol. Cell. Biol. 25: 4727-4741 (2005). (Immunocytochemistry) 15. Establishment and characterization of SRIK-NKL: a novel CD8+ natural killer/T cell line derived from a patient with leukemic phase of acute lymphoblastic lymphoma. Bejai I.S. Srivastava and Maya D. Srivastava. Leukemia Research, 29 (7): 771-783 (2005). 16. Immunology of Cutaneous Vasculitis Associated with both Etanercept and Infliximab. Srivastava, M. D., Alexander, F. & Tuthill, R. J. Scandinavian Journal of Immunology, 61 (4): 329-336 (2005). 17. An association of DNMT3b protein expression with P16INK4a promoter hypermethylation in non-smoking female lung cancer with human papillomavirus infection. Torng-Sen Lin, Huei Lee, Ruei-A Chend, Ming-Lin Ho, Chia-Yu Lin, Ya-Hui Chen, Yi Yu Tsai, Ming-Chih Chou, and Ya-Wen Cheng. Cancer Letters, 226 (1): 77-84 (2005). Imgenex antibodies cited: 1. Dnmt1 (IMG-261A) [IHC-P, Fig.1 (lung tumors)]. 18. Stage-specific induction of DNA methyltransferases in olfactory receptor neuron development. Jessica L. MacDonald, Christopher S.Y. Gin and A. Jane Roskams. Developmental Biology, In Press, Corrected Proof, Available online 8 November 2005. (IHC on paraffin) 19. Zebrafish Dnmt1 and Suv39h1 regulate organ-specific terminal differentiation during development. Kunal Rai, Lincoln D. Nadauld, Stephanie Chidester, Elizabeth J. Manos, Smitha R. James, Adam R. Karpf, Bradley R. Cairns, and David A. Jones. Mol. Cell. Biol., 26: 7077-7085 (2006). (Zebra fish) 20. DNA methyltransferase expression in the human endometrium: down-regulation by progesterone and estrogen. Yamagata Y, A Asada, I Tamura, L Lee, R Maekawa, K Taniguchi, T Taketani, A Matsuoka, H Tamura, N Sugino. Human Reproduction 24:1126-1132 (2009). Imgenex antibodies cited: 1. IMG-261A (dnmt1): IHC(parafin), human endometrium tissue, Fig. 1A. 2. IMG-184A (dnmt3b): IHC(parafin), human endometrium tissue, Fig. 1B. 21. Role of the Polycomb Repressive Complex 2 in Acute Promyelocytic Leukemia. Raffaella Villa, Diego Pasini, Arantxa Gutierrez, Lluis Morey, Manuela Occhionorelli, ,et al. Cancer Cell, 11 (6): 513-525 (2007). 22. Neither DNA hypotheylation nor changes in the kinetics of erythroid differentiation explain 5-azacytidine's ability to induce human fetal hemoglobin. Mabaera R, M Greene, C Richardson, S Conine, C Kozul, C Lowrey. Blood 111:411-420 (2008). WB (human primary erythroid cells): Figs. 5C, 6B; The antibody is DNMT1 siRNA validated in both figures.
23. Aberrant epigenetic modifications in hepatocarcinogenesis induced by Hepatitis B virus X protein. Park Y, B Sohn, E Yu, D Suh, Y Chung, J Lee, S Surzychki, Y Lee. Gastroenterology 132: 1476-1494 (2007). Imgenex products cited (Chang liver and Hep G2 cell lines): 1. IMG-261(DNMT1): WB, Figs. 1A, 4A,B. 2. IMG-268A (DNMT3A): WB, ChIP, Figs. 1B, 2B, 4A,B. 24. Epigenetic silencing of O6 -methlyguanin-DNA methyltransferase gene in NiS-transformed cells. Weidong J, L Yang, L Yu, J Yuan, D Hu, W Zhang, J Yang, Y Pang, W Li, J Lu, J Fu, J Chen, Z Lin, W Chen, Z Zhuang. Carcinogenesis doi:10.1093/carcin/bgn012. (2008). ChIP (human bronchial epethial cell line 16HBE cells), Fig. 6 A,B. 25. CTCF activates PARP-1 affecting DNA methylation machinery. Guastafierr T, B Cecchinelli, M Zampieri, A Reale, G Riggo, O Sthandier, G Zupi, L Calabrese, P Caiafa. JBC 10.1074/jbc.M801170200 (2008). WB (L929 mouse fibroblasts and A1 mouse embryonic fibroblasts), Figs. 2A-E; 4A,C; 5. 26. Helicobacter pylori regulates p21WAF1 by histone H4 acetylation. Xia G,R Schneider-Stock, A Diestel, C Habold, S Krueger, A Roessner, M Naumann, U Lendeckel. BBRC 369: 526-531 (2008). IMGENEX products cited (human NCI N87 gastric carcinoma cells): WB (Fig 1) 1. Dnmt1 (IMG-261) 2. Dnmt3a (IMG-268) 3. Dnmt3b (IMG-184) 27. CCCTC-binding factor activates PARP-1 affecting DNA methylation machinery. Guastafierro T, B Cecchinelli, M Zampieri, A Reale, G Riggio, O Sthandier, G Zupi, L Calabrese, P Caiafa. JBC 283: 21873-71880 (2008). WB: (mouse fibroblasts), Figs. 2A, 4A; IP: (mouse fibroblasts), Fig. 2E. 28. Breast cancer-associated fibroblasts confer AKT1-mediated epigenetic silencing of Cystatin M in epithelial cells. Lin H, T Zuo, C Lin, C Kuo, S Liyanarachchi, S Sun, R Shen, D Deatherage, D Potter, L Asamoto, S Lin, P Yan, A Cheng, M Ostrowski, T Huang. Cancer Research 68: 10257-10266 (2008). ChIP (human breast cancer cell line MCF10A), Fig. 5A. 29. Parp1 localizes within the Dnmt1 promoter and protects its unmethylated state by its enzymatic activity. Zampieri M, C Passananti, R Calabrese, M Perilli, N Corbi, F De Cave, T Guastafierro, M Bacalini, A Reale, G Amicosante, L Calabrese, J Zlatanova, P Caifa. PLoS ONE 4: e4717 (2009). L929 mouse fibroblasts: WB (Figs 2A, 5B), IP (Fig 4D). 30. Epigenetic silencing of CCAAT/enhancer-binding protein ∂ activity by YY1/polycomb group/DNA methlytransferase complex. Ko C, H Hsu, M Shen, W Chang, J Wang. JBC 283: 30919-30932 (2008). Imgenex antibodies cited for HeLa cells in Fig. 7A (IF), 7B (WB following a DNA Affinity Precipitation Assay of nuclear extracts), 7C and D (ChIP): . 1. IMG-261A (Dnmt1) 2. IMG-268A (Dnmt3a) 3. IMG-184A (Dnmt3b). 31. DNA cytosine methylation in the bovine leukemia virus promoter is associated with latency in a lymphome derived B cell line. Potential involvement of direct inhibition of cAMP responsive element (CRE) binding protein/CRE modulator/activation transcription factor binding. Pierard V, A Guigen, L Colin, G Wijmeersch, C Vanhulle, B Driessche, A Dekoninck, J Blazkova, C Cardona, M Merimi, V Vierendeel, C Calomme, T Nguyen, M Nuttinck, J Twizere, R Kettmann, D Portetelle, A Burny, I Hirsch, O Rohr, C Lint. JBC 285:19436-19449 (2010): The following IMGENEX products were used in western blot: DNMT1 (IMG-261A), DMNT3A (IMG-268A), DNMT3B (IMG-184A) in Fig 8: Nuclear extracts from ovine (sheep) cell lines L267 and L267LTaxSN. L267 is a B-cell lymphoma cell line established from Bovine Leukemia Virus (BLV) infected sheep. L267LTaxSN is a derivative of L267, see the publication for additional details. 32. The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression. Achour M, X Jacq, P Ronde, M alhosin, C Charlot, T Chataigneau, M Jeanblanc, M Maculuso, A Giodano, AD Hughes, VB Schini-Kerth, C Bronner. Oncogene 27:2187-2197 (2008).
Note: DNMT1 was detected at ~180 kDa. Proliferating Jurkat cell cultures had a higher DNMT1 expression level than confluent Jurkat cells cultures (Fig 3a). w
Note: The specificity of the DNMT1 (IMG-261A) was siRNA validated in Fig 7a by WB in Jurkat. Cells treated with DNMT1 siRNA, but not scrambled siRNA, showed a dramatic reduction of DNMT1 expression.
Research purposes only.
Not for diagnostic or in vivo use. This product is guaranteed to perform as indicated on the datasheet for one year from the date of purchase.
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