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93140
DNMT3A Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

DNMT3A Antibody Sampler Kit #93140

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DNMT3A Antibody Sampler Kit: Image 1

Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either DNMT3A (E9P2F) Rabbit mAb or DNMT3A (D2H4B) Rabbit mAb #32578, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across HUWE1, a known target gene of DNMT3A (see additional figures containing ChIP-qPCR data).

DNMT3A Antibody Sampler Kit: Image 2

Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using DNMT3A (D23G1) Rabbit mAb.

DNMT3A Antibody Sampler Kit: Image 3

Confocal immunofluorescent analysis of NCCIT cells (left, positive) and HCT 116 cells (right, negative) using DNMT3A Isoform 2 (E1Y5O) Rabbit mAb (green). Actin filaments were labeled with Alexa Fluor® 647 Phalloidin (red) #8940. Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

DNMT3A Antibody Sampler Kit: Image 4

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

DNMT3A Antibody Sampler Kit: Image 5

Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either DNMT3A (E9P2F) Rabbit mAb #49768 or DNMT3A (D2H4B) Rabbit mAb #32578, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across HUWE1 (upper) and ARHGEF2 (lower), known target genes of DNMT3A (see additional figures containing ChIP-qPCR data).

DNMT3A Antibody Sampler Kit: Image 6

Immunohistochemical analysis of paraffin-embedded SHSY5Y cell pellet (left, high), HCT116 cell pellet (middle, moderate) and T47D cell pellet (right, low) using DNMT3A (D23G1) Rabbit mAb.

DNMT3A Antibody Sampler Kit: Image 7

Immunoprecipitation of DNMT3A isoform 2 (DNMT3A2) protein from NCCIT cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is DNMT3A Isoform 2 (E1Y5O) Rabbit mab. Western blot analysis was performed using DNMT3A Isoform 2 (E1Y5O) Rabbit mAb. Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) #93702 was used as the secondary antibody.

DNMT3A Antibody Sampler Kit: Image 8

Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either

DNMT3A (E9P2F) Rabbit mAb 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 HUWE1 Intron 26 Control Primers #33770, human ARHGEF2 intron 13 primers, human SDHAP1 intron 10 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.

DNMT3A Antibody Sampler Kit: Image 9

Immunohistochemical analysis of paraffin-embedded human placenta using DNMT3A (D23G1) Rabbit mAb.

DNMT3A Antibody Sampler Kit: Image 10

Western blot analysis of extracts from NCCIT, NTERA-2 cl.D1, and HCT 116 cells using DNMT3A Isoform 2 (E1Y5O) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). As expected, HCT 116 cells are negative for DNMT3A isoform 2 (DNMT3A2) expression.

DNMT3A Antibody Sampler Kit: Image 11

Immunoprecipitation of DNMT3A from NCCIT cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is DNMT3A (E9P2F) Rabbit mAb. Western blot analysis was performed using DNMT3A (E9P2F) Rabbit mAb.

DNMT3A Antibody Sampler Kit: Image 12

Immunohistochemical analysis of paraffin-embedded human testis using DNMT3A (D23G1) Rabbit mAb.

DNMT3A Antibody Sampler Kit: Image 13

Western blot analysis of extracts from various cell lines using DNMT3A (E9P2F) Rabbit mAb.

DNMT3A Antibody Sampler Kit: Image 14

Western blot analysis of extracts from 293, NIH/3T3 and H-4-II-E cells using DNMT3A (D23G1) Rabbit mAb.

To Purchase # 93140T
Product # Size Price
93140T
1 Kit  (3 x 20 µl) N/A

Product Description

The DNMT3A Antibody Sampler Kit provides an economical means of detecting various isoforms of DNMT3A, including isoform 1 (DNMT3A1) and isoform 2 (DNMT3A2). The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Each antibody in the DNMT3A Antibody Sampler Kit detects endogenous levels of its target protein. DNMT3A (E9P2F) Rabbit mAb detects endogenous levels of total DNMT3A protein, including isoform 1 (DNMT3A1) at 130 kDa and isoform 2 (DNMT3A2) at 95 kDa. This antibody also detects an additional isoform of DNMT3A protein at 85 kDa. DNMT3A (D23G1) Rabbit mAb only detects endogenous levels of DNMT3A1 at 130 kDa. DNMT3A Isoform 2 (E1Y5O) Rabbit mAb only detects endogenous levels of DNMT3A2 at 95 kDa. These antibodies do not cross-react with other DNMT proteins, including DNMT1 and DNMT3B.

Source / Purification

DNMT3A (E9P2F) Rabbit mAb is produced by immunizing animals with recombinant protein specific to the carboxy terminus of human DNMT3A isoform 1 (DNMT3A1). DNMT3A (D23G1) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to residue Gly91 of human DNMT3A1. DNMT3A Isoform 2 (E1Y5O) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human DNMT3A isoform 2 (DNMT3A2) 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 over-expressed in many cancers, including acute and chronic myelogenous leukemias, in addition to colon, breast and stomach carcinomas (9-12).

There are at least two protein isoforms expressed from the DNMT3A locus, DNMT3A isoform 1 (DNMT3A1) and DNMT3A isoform 2 (DNMT3A2). DNMT3A2 is expressed from an intronic promoter that is downstream from the DNMT3A1 promoter (13,14). As a result, the N-terminal 223 amino acids found in DNMT3A1 are replaced by a different 24 amino acid found in DNMT3A2. Although they have distinct N-termini, both isoforms contain the PWWP domain required for binding to tri-methylated histone H3 lysine 36 (H3K36me3) and the ADD domain required for histone binding and transcriptional regulation. DNMT3A1 is lowly expressed in most cell and tissue types and is localized to heterochromatic regions. DNMT3A2 expression appears to be developmentally regulated and limited to embryonic stem cells, where it is localized to euchromatic regions of the genome, suggesting distinct functions for DNMT3A1 and DNMT3A2. DNMT3A2 is the predominant isoform responsible for de novo DNA methylation in embryonic stem cells. In addition, DNMT3A2 is mutated and/or highly expressed in a number of different cancers, including acute myeloid leukemia (AML), teratocarcinoma, neuroblastoma, and lung, testicular, gastric, and breast cancer (13-17).

  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.
  13. Gujar, H. et al. (2019) Genes (Basel) 10, pii: E172. doi: 10.3390/genes10020172.
  14. Chen, B.F. and Chan, W.Y. (2014) Epigenetics 9, 669-77.
  15. Chen, B.F. et al. (2014) Epigenetics 9, 119-28.
  16. Gao, J. et al. (2013) J Exp Clin Cancer Res 32, 86.
  17. Yu, Z. et al. (2015) Mol Carcinog 54, 707-19.

Pathways & Proteins

Explore pathways + proteins related to this product.

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's 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's 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.

For Research Use Only. Not For Use In Diagnostic Procedures.
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