Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

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

33D3   5-mC   5-methylcytosine   cytosine   DIP   DIP kit   DNA IP   DNA methylation   DNA-IP   MeDIP   methyl-cytidine   methyl-cytosine   methyl-DNA   Methyl-DNA IP   methylcytosine  

No. Size Price
28692S 100 µl ( 10 immunoprecipitations ) ¥3,100.00 现货查询 购买询价
28692 carrier free & custom formulation / quantityemail request
Applications Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
IF-IC 1:1600 All Species Expected, Endogenous Rabbit IgG
Dot Blot 1:1000
MeDIP 1:50

Species cross-reactivity is determined by western blot.

Applications Key: IF-IC=Immunofluorescence (Immunocytochemistry), Dot Blot=DNA Dot Blot, MeDIP=Methylated DNA IP,

Specificity / Sensitivity

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.

Source / Purification

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

MeDIP

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.

MeDIP

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.

Dot Blot-DNA

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.

ELISA-DNA Oligo

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.

MeDIP

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.

Dot Blot-DNA

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.

IF-IC

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).

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).

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, 2571-87.
  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. 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.

Application References

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

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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

XP is a registered 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.

Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.

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