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Santa Cruz discontinued a large number of its polyclonal products as a result of the USDA settlement that was made public May 19th 2016

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Endogenous Rabbit IgG

Confocal immunofluorescent analysis of 293T cells transfected with a construct expressing DYKDDDDK-tagged TET1 catalytic domain (TET1-CD) using 5-Carboxylcytosine (5-caC) (D7S8U) Rabbit mAb (green) and DYKDDDDK Tag (9A3) Mouse mAb #8146 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). As expected, 293T cells expressing TET1-CD (red) exhibit increased levels of 5-carboxylcytosine (green).

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The specificity of 5-Carboxylcytosine (5-caC) (D7S8U) Rabbit mAb was determined by ELISA. The antibody was titrated against a single-stranded DNA oligo containing either unmodified cytosine or differentially modified cytosine (5-mC, 5-hmC, 5-caC, 5-fC). As shown in the graph, the antibody only binds to the oligo containing 5-caC.

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Specificity of various modified cytosine antibodies were determined by dot blot. The same sequence of a 387-base pair DNA fragment was generated by PCR using exclusively unmodified cytosine, 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-carboxylcytosine (5-caC), or 5-formylcytosine (5-fC). The respective DNA fragments were blotted onto a nylon membrane, UV cross-linked, and probed with 5-Carboxylcytosine (5-caC) (D7S8U) Rabbit mAb #36836, 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692, 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb #51660, and 5-Formylcytosine (5-fC) (D5D4K) Rabbit mAb #74178. As shown, the 5-Carboxylcytosine (5-caC) (D7S8U) Rabbit mAb shows specificity for 5-caC.

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5-Carboxylcytosine (5-caC) (D7S8U) Rabbit mAb specificity was determined by DNA immunoprecipitation. DNA IPs were performed with genomic DNA prepared from human HCT 116 cells, spiked with DNA containing either unmodified cytosine, 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-carboxylcytosine (5-caC), or 5-formylcytosine (5-fC). IPs were performed using 5-Carboxylcytosine (5-caC) (D7S8U) Rabbit mAb and the SimpleDIP™ Methylated DNA IP (MeDIP) Kit #76853. The enriched DNA was quantified by real-time PCR using primers specific to the spiked-in control DNA sequence. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input DNA, which is equivalent to one.

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Product Usage Information

Application Dilutions
Immunofluorescence (Immunocytochemistry) 1:200
DNA Dot Blot 1:1000

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.

Specificity / Sensitivity

5-Carboxylcytosine (5-caC) (D7S8U) Rabbit mAb recognizes endogenous levels of 5-carboxylcytosine. This antibody has been validated using ELISA, dot blot, and synthetic spike-in DNA MeDIP assays and shows high specificity for 5-carboxylcytosine.

Source / Purification

Monoclonal antibody is produced by immunizing animals with 5-carboxylcytidine.

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).

TET protein-mediated cytosine hydroxymethylation was initially demonstrated in mouse brain and embryonic stem cells (5, 8). Since then this modification has been discovered in many tissues, with the highest levels found in the brain (9). While 5-fC and 5-caC appear to be short-lived intermediate species, there is mounting evidence showing that 5-hmC is a distinct epigenetic mark with various unique functions (10,11). The modified base itself is stable in vivo and interacts with various readers including MeCP2 (11,12). The global level of 5-hmC increases during brain development, and 5-hmC is enriched at promoter regions and poised enhancers. Furthermore, there is an inverse correlation between levels of 5-hmC and histone H3K9 and H3K27 trimethylation, suggesting a role for 5-hmC in gene activation (12). Lower amounts of 5-hmC have been reported in various cancers including myeloid leukemia and melanoma (13,14).

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.  Kriaucionis, S. and Heintz, N. (2009) Science 324, 929-30.

9.  Globisch, D. et al. (2010) PLoS One 5, e15367.

10.  Gao, Y. et al. (2013) Cell Stem Cell 12, 453-69.

11.  Mellén, M. et al. (2012) Cell 151, 1417-30.

12.  Wen, L. et al. (2014) Genome Biol 15, R49.

13.  Delhommeau, F. et al. (2009) N Engl J Med 360, 2289-301.

14.  Lian, C.G. et al. (2012) Cell 150, 1135-46.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SimpleDIP is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
Tween is a registered trademark of ICI Americas, Inc.

5-Carboxylcytosine (5-caC) (D7S8U) Rabbit mAb