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

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

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
All Endogenous Rabbit IgG
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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Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

Stock Solutions

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. Aspirate media, cover cells completely with ice-cold 70% ethanol.
  2. Allow cells to fix for 5 minutes at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
  4. Add 1.5 M HCl and incubate for 30 minutes at room temperature.
  5. Aspirate HCl and rinse two times in 1X PBS for 5 minutes each.
  6. Proceed with Immunostaining section C.

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 minutes.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 minutes each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
  7. Rinse three times in 1X PBS for 5 minutes each.
  8. Mount samples in an appropriate antifade reagent such as Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold AntiFade Reagent with DAPI (#8961).
  9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted December 2015

Protocol Id: 864

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, 2571-87.
  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.
For Research Use Only. Not For Use In Diagnostic Procedures.

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