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.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 6
#5032

DNMT1 (D63A6) XP® Rabbit mAb

Western Blotting Image 1: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from 293 and F9 cells using DNMT1 (D63A6) XP® Rabbit mAb.
Immunohistochemistry Image 1: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human ovarian serous adenocarcinoma using DNMT1 (D63A6) XP® Rabbit mAb.
Immunohistochemistry Image 2: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human soft tissue squamous cell carcinoma using DNMT1 (D63A6) XP® Rabbit mAb.
Immunohistochemistry Image 3: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded normal human thymus using DNMT1 (D63A6) XP® Rabbit mAb.
Immunohistochemistry Image 4: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded normal human testis using DNMT1 (D63A6) XP® Rabbit mAb.
Immunohistochemistry Image 5: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded normal human lymph node using DNMT1 (D63A6) XP® Rabbit mAb.
Immunohistochemistry Image 6: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded normal human placenta using DNMT1 (D63A6) XP® Rabbit mAb (left) or a DNMT1 Rabbit mAb (right). These two antibodies detect unique, non-overlapping epitopes on human DNMT1. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.
Immunohistochemistry Image 7: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using DNMT1 (D63A6) XP® Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right). 
Immunohistochemistry Image 8: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded HCT 116 cell pellet, untreated (left) or Doxycycline-treated (10 ng/mL, 72 hr, right), using DNMT1 (D63A6) XP® Rabbit mAb.
Immunofluorescence Image 1: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
Confocal immunofluorescent analysis of COS cells using DNMT1 (D63A6) XP® Rabbit mAb (green). Actin filaments were labeled using DY-554 phalloidin (red).
Flow Cytometry Image 1: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
Flow cytometric analysis of COS7 cells using DNMT1 (D63A6) XP® Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L) F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: DNMT1 (D63A6) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT116 wild type cells (left panel) and HCT116 DNMT3B/DNMT1 double knockout cells (right panel), both treated with doxorubicin (1 uM for 24 hrs), and either DNMT1 (D63A6) XP® Rabbit mAb #5032 or Normal Rabbit IgG #2729, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR, using human POMC promoter primers and human Stat2 promoter primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.