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84884
Human Reactive DNA Demethylation Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit
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Human Reactive DNA Demethylation Antibody Sampler Kit #84884

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Western blot analysis of extracts from DLD-1 or 293T cells, either wild type (+/+) or TET2 knockout (-/-), using TET2 (D6B9Y) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). As expected, DLD-1 and 293T (+/+) cells are positive for TET2 expression, while TET2 knockout (-/-) cells are negative.
Western blot analysis of extracts from control 293T cells (lane 1) or CRISPR/Cas9 TET1 knockdown (KD) 293T cells (lane 2) using TET1 (E5F1O) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). SDS-PAGE was performed using a 3-8% Tris-acetate gel.
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.
Western blot analysis of extracts from HEL 92.1.7, IMR-32, and MCF7 cells using TET3 (E2S3C) Rabbit mAb.
Western blot analysis of extracts from various cell lines using TDG (E5T5G) Rabbit mAb.
Chromatin immunoprecipitations were performed with cross-linked chromatin from 293T cells and either TET2 (D6B9Y) 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 ZNF335 Promoter Primers #25946, human TAF12 exon1 primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. 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.
Western blot analysis of extracts from various cell lines using TET1 (E5F1O) Rabbit mAb. SDS-PAGE was performed using a 3-8% Tris-acetate gel.
Immunoprecipitation of TET3 protein from HEL 92.1.7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is TET3 (E2S3C) Rabbit mAb. Western blot analysis was performed using TET3 (E2S3C) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody. Immunoprecipitation with primary antibody was performed for two hours at 4°C.
Western blot analysis of extracts from HCT 116 cells, transfected with control siRNA (-) or TDG siRNA (+), using TDG (E5T5G) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from mES and COS-7 cells using TET1 (E5F1O) Rabbit mAb. SDS-PAGE was performed using a 3-8% Tris-acetate gel.
Western blot analysis of extracts from A20 and BA/F3 cells using TDG (E5T5G) Rabbit mAb.
Chromatin immunoprecipitations were performed with cross-linked chromatin from mES cells and either TET1 (E5F1O) 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® Mouse Oct-4 Promoter Primers #4653, mouse Xist intron1 primers, and mouse Nanog 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.

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To Purchase # 84884
Cat. # Size Qty. Price
84884T		 1 Kit  (4 x 20 microliters)

Bulk & Custom Formulation

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
TET1 (E5F1O) Rabbit mAb 40142 20 µl
  • WB
  • ChIP
 H M Mk 300 Rabbit IgG
TET2 (D6B9Y) Rabbit mAb 18950 20 µl
  • WB
  • IP
  • ChIP
 H 280 Rabbit IgG
TET3 (E2S3C) Rabbit mAb 85016 20 µl
  • WB
  • IP
 H Mk 235 Rabbit IgG
TDG (E5T5G) Rabbit mAb 99105 20 µl
  • WB
 H M R Mk 58, 60 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
 Goat 

Product Description

The Human Reactive DNA Demethylation Antibody Sampler Kit provides an economical means of detecting TET protein family members and TDG protein. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Background

Methylation of DNA at cytosine residues is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting, and mammalian development (1,2). 5-methylcytosine is a repressive epigenetic mark established de novo by two enzymes, DNMT3a and DNMT3b, and is maintained by DNMT1 (3,4). 5-methylcytosine was originally thought to be passively depleted during DNA replication. However, subsequent studies have shown that Ten-Eleven Translocation (TET) proteins TET1, TET2, and TET3 can catalyze the oxidation of methylated cytosine to 5-hydroxymethylcytosine (5-hmC) (5). Additionally, TET proteins can further oxidize 5-hmC to form 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC), both of which are excised by thymine-DNA glycosylase (TDG), effectively linking cytosine oxidation to the base excision repair pathway and supporting active cytosine demethylation (6,7). TET1 is highly expressed in embryonic stem cells and is essential for maintaining stem cell pluripotency (8). Aberrant TET1 expression has also been implicated in a variety of cancers, including hepatocellular carcinoma, T-cell acute lymphoblastic leukemia (T-ALL), and triple-negative breast cancer (TNBC), among others (9-11). TET2 is frequently mutated in myeloid dysplastic syndrome (MDS) and diffuse large B-cell lymphomas (12,13). TET2 protein expression is often reduced in solid tumors such as prostate cancer, melanoma, and oral squamous cell carcinoma (14-16). TET3 plays key roles in regulating early development and neonatal growth (17,18). TET2/TET3 deficiency can lead to myeloid cell, B cell, and invariant natural killer T (iNKT) cell malignancies. In Tregs, TET2/TET3 deficiency in mice leads to hyperproliferation and inflammatory disease (19,20). Knockout or catalytic inactivation of TDG leads to embryonic lethality (21,22). SUMOylation of TDG has been reported to help it dissociate from its abasic product, thereby increasing catalytic turnover (23). Additional studies suggest that SUMOylation affects TDG’s cellular localization or lowers its base excision activity, allowing it to act as a ‘reader’ protein for 5-fC and 5-caC modified DNA (24).

  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. Okano, M. et al. (1999) Cell 99, 247-57.
  4. Li, E. et al. (1992) Cell 69, 915-26.
  5. Tahiliani, M. et al. (2009) Science 324, 930-5.
  6. He, Y.F. et al. (2011) Science 333, 1303-7.
  7. Ito, S. et al. (2011) Science 333, 1300-3.
  8. Ito, S. et al. (2010) Nature 466, 1129-33.
  9. Shirai, K. et al. (2021) Cancer Sci 112, 2855-2869.
  10. Bamezai, S. et al. (2021) Leukemia 35, 389-403.
  11. Good, C.R. et al. (2018) Cancer Res 78, 4126-4137.
  12. Langemeijer, S.M. et al. (2009) Nat Genet 41, 838-42.
  13. Asmar, F. et al. (2013) Haematologica 98, 1912-20.
  14. Nickerson, M.L. et al. (2013) Hum Mutat 34, 1231-41.
  15. Lian, C.G. et al. (2012) Cell 150, 1135-46.
  16. Jäwert, F. et al. (2013) Anticancer Res 33, 4325-8.
  17. Peat, J.R. et al. (2014) Cell Rep 9, 1990-2000.
  18. Tsukada, Y. et al. (2015) Sci Rep 5, 15876.
  19. Nakatsukasa, H. et al. (2019) Int Immunol 31, 335-347.
  20. Yue, X. et al. (2019) Nat Commun 10, 2011.
  21. Cortellino, S. et al. (2011) Cell 146, 67-79.
  22. Cortázar, D. et al. (2011) Nature 470, 419-23.
  23. Hardeland, U. et al. (2002) EMBO J 21, 1456-64.
  24. Coey, C.T. and Drohat, A.C. (2018) Nucleic Acids Res 46, 5159-5170.

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