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28692
5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb
MeDIP Kits & Reagents

5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692

MeDIP

DNA immunoprecipitations were performed with 1 μg of genomic DNA from NCCIT cells and either 10 μl of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692 or 10 μl of Rabbit (DA1E) mAb IgG XP® Isotype Control (DIP Formulated) #75708 using SimpleDIP™ Methylated DNA IP (MeDIP) Kit #76853. The enriched DNA was quantified by real-time PCR using human Aqp2 intron 5 primers, human TIMP3 promoter primers, SimpleDIP™ Human Testis-Specific H2B Promoter Primers #65822, 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 DNA, which is equivalent to one.

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MeDIP

DNA immunoprecipitations were performed with 1 μg of genomic DNA from mouse embryonic stem cells and either 10 μl of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692 or 10 μl of Rabbit (DA1E) mAb IgG XP® Isotype Control (DIP Formulated) #75708 using SimpleDIP™ Methylated DNA IP (MeDIP) Kit #76853. The enriched DNA was quantified by real-time PCR using mouse Aqp2 exon 1 primers, SimpleDIP™ Mouse Intracisternal-A Particle (IAP) LTR Primers #74803, mouse Lamc3 exon 1 primers, and SimpleChIP® Mouse GAPDH Intron 2 Primers #8986. 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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IF-IC

Confocal immunofluorescent analysis of 293T cells transfected with a construct expressing DYKDDDDK-tagged TET1 catalytic domain (TET1-CD) using 5-Methylcytosine (5-mC) (D3S2Z) 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 decreased levels of 5-methylcytosine (green).

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Dot Blot-DNA

DNA fragments from HCT 116 wild type (WT) and DNMT1/DNMT3B knock-out (KO) cells were blotted onto a nylon membrane, UV cross-linked, and probed with 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb. The top panel shows the antibody detecting more methylated cytosine in the wild type cells, while the bottom panel shows the membrane stained with methylene blue.

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Dot Blot-DNA

The specificity of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb was 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-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb. The top panel shows the antibody only binding to the DNA fragment containing 5-mC, while the bottom panel shows the membrane stained with methylene blue.

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ELISA-DNA Oligo

The specificity of 5-Methylcytosine (5-mC) (D3S2Z) 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-mC.

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MeDIP

The specificity of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb was determined by DNA immunoprecipitations. DNA IPs were performed with genomic DNA prepared from mouse embryonic stem cells, spiked with control DNA containing either unmethylated cytosine, 5-methylcytosine (5-mC), or 5-hydroxymethylcytosine (5-hmc). IPs were performed using 5-Methylcytosine (5-mC) (D3S2Z) 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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Application Dilutions
Immunofluorescence (Immunocytochemistry) 1:1600
DNA Dot Blot 1:1000
Methylated DNA 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.

5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb recognizes endogenous levels of 5-methylcytosine. This antibody has been validated using ELISA, dot blot, and MeDIP assays and shows high specificity for 5-methylcytosine.

Species Reactivity:

All Species Expected

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

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).

Normally DNA methylation occurs in a bimodal fashion, such that CpG dinucleotides are largely methylated across the genome, except in short stretches of CpG-rich sequences associated with gene promoters, known as CpG-islands, where methylation is virtually absent (8). Cancer cell genomes often undergo global hypomethylation, while CpG-islands become hypermethylated, causing their associated promoters to become repressed (9). There is evidence that a number of aberrantly hypermethylated CpG-islands found in carcinomas occur at tumor suppressor genes such as RB1, MLH1, and BRCA1 (10).

  1. Hermann, A. et al. (2004) Cell. Mol. Life Sci. 61, 2571-2587.
  2. Turek-Plewa, J. and Jagodziński, P.P. (2005) Cell. Mol. Biol. Lett. 10, 631-647.
  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. Suzuki, M.M. and Bird, A. (2008) Nat Rev Genet 9, 465-76.
  9. Berman, B.P. et al. (2012) Nat Genet 44, 40-6.
  10. Sproul, D. and Meehan, R.R. (2013) Brief Funct Genomics 12, 174-90.
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
SimpleDIP is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.

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