Revision 4
#72335
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For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:
W, 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 Usage Information
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
Specificity/Sensitivity
DNMT3B (E2Q3Z) Rabbit mAb recognizes endogenous levels of total DNMT3B protein. This antibody does not cross react with DNMT3A or DNMT1.
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).
Background References
- Hermann, A. et al. (2004) Cell. Mol. Life Sci. 61, 2571-87.
- Turek-Plewa, J. and Jagodziński, P.P. (2005) Cell. Mol. Biol. Lett. 10, 631-47.
- Kim, G.D. et al. (2002) EMBO J. 21, 4183-95.
- Fuks, F. et al. (2001) EMBO J. 20, 2536-44.
- Geiman, T.M. et al. (2004) Biochem. Biophys. Res. Commun. 318, 544-55.
- Rountree, M.R. et al. (2000) Nat. Genet. 25, 269-77.
- Pradhan, S. and Kim, G.D. (2002) EMBO J. 21, 779-88.
- Fuks, F. et al. (2003) Nucleic Acids Res. 31, 2305-12.
- Mizuno, S. et al. (2001) Blood 97, 1172-9.
- Robertson, K.D. et al. (1999) Nucleic Acids Res. 27, 2291-8.
- Xie, S. et al. (1999) Gene 236, 87-95.
- 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
W: 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 G: Goat 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.
Limited Uses
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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 4
#72335
DNMT3B (E2Q3Z) Rabbit mAb
Western blot analysis of extracts from K-562, Huh7, and COS-7 cell lines using DNMT3B (E2Q3Z) Rabbit mAb.
Immunoprecipitation of DNMT3B from NCCIT cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is DNMT3B (E2Q3Z) Rabbit mAb. Western blot analysis was performed using DNMT3B (E2Q3Z) Rabbit mAb and Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127.
Flow cytometric analysis of NCCIT cells (green), HCT116 WT cells (red), or HCT116 DNMT3B KO cells (blue), using DNMT3A (E2Q3Z) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines).Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Revision 4
#72335
DNMT3B (E2Q3Z) Rabbit mAb
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either DNMT3B (E2Q3Z) Rabbit mAb #72335 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 SimpleChIP® Human EIF4G1 Intron 8 Primers #29118, human CROCC intron 1 primers, and SimpleChIP® Human GAPDH Promoter Primers #4471. 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.
CUT&RUN was performed with NCCIT cells and either DNMT3B (E2Q3Z) Rabbit mAb or DNMT3B (E8A8A) XP® Rabbit mAb #57868, using CUT&RUN Assay Kit #86652. DNA libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figures show binding across HUWE1, a known target gene of DNMT3B (see additional figures containing CUT&RUN-qPCR data).
CUT&RUN was performed with NCCIT cells and either DNMT3B (E2Q3Z) Rabbit mAb or DNMT3B (E8A8A) XP® Rabbit mAb #57868, using CUT&RUN Assay Kit #86652. DNA libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across HUWE1 (upper) and SMARCA1 (lower), known target genes of DNMT3B (see additional figures containing CUT&RUN-qPCR data).
Revision 4
#72335
DNMT3B (E2Q3Z) Rabbit mAb
CUT&RUN was performed with NCCIT cells and either DNMT3B (E2Q3Z) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human HUWE1 intron 26 primers, human ARHGEF2 promoter primers, and SimpleChIP® Human GAPDH Exon 1 Primers #5516. 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.