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36836
5-Carboxylcytosine (5-caC) (D7S8U) Rabbit mAb
Primary Antibodies

5-Carboxylcytosine (5-caC) (D7S8U) Rabbit mAb #36836

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
All Transfected Only Rabbit IgG
IF-IC

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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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) 1:200
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.

5-Carboxylcytosine (5-caC) (D7S8U) Rabbit mAb detects 5-caC by IF in cells over-expressing the TET1 catalytic domain and by dot blot using double-stranded PCR fragments containing 5-caC. Many cells and tissues contain very low endogenous levels of 5-caC that may fall below the detection limits of this antibody. This antibody has been validated using ELISA, dot blot, and synthetic spike-in DNA MeDIP assays and shows high specificity for 5-caC.

Species Reactivity:

All Species Expected

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.

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