Confocal immunofluorescent analysis of 293T cells transfected with a construct expressing DDK-tagged TET1 catalytic domain (TET1-CD) using 5-Formylcytosine (5-fC) (D5D4K) 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-formylcytosine (green).
Learn more about how we get our imagesThe specificity of 5-Formylcytosine (5-fC) (D5D4K) 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-Formylcytosine (5-fC) (D5D4K) Rabbit mAb. The upper panel shows the antibody only binding to the DNA fragment containing 5-fC, while the lower panel shows the membrane stained with methylene blue.
Learn more about how we get our images5-Formylcytosine (5-fC) (D5D4K) Rabbit mAb specificity 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-fC.
Learn more about how we get our imagesNOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100):
To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton™ X-100.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
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.
posted December 2015
Note: This protocol is written for spotting fragmented, purified genomic DNA (titration of 1000 ng, 500 ng, 250 ng, 125 ng, 62.5 ng, 31.25 ng, and 15.625 ng) onto a positively charged nylon membrane using a 96-well dot blotting apparatus. Depending on the source and type of DNA, more or less DNA may be required for detection with the antibody.
Before Starting:
• Purify genomic DNA using a genomic DNA purification protocol or kit and sonicate
genomic DNA to generate fragments between 200 and 500 bp. DNA fragment size
can be analyzed by gel electrophoresis on a 1% agarose gel with a 100 bp DNA
marker.
• Cut a piece of nylon membrane to the size of the dot blot manifold.
• Wet nylon membrane with 10x SSC Buffer.
• Dry membrane by placing it in 96-well dot blot apparatus and applying vacuum.
NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately following incubation and declines over the following 2 hr
posted November 2015
Application | Dilutions |
---|---|
Immunofluorescence (Immunocytochemistry) | 1:200 |
DNA Dot Blot | 1:1000 |
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-Formylcytosine (5-fC) (D5D4K) Rabbit mAb detects 5-fC by IF in cells over-expressing the TET1 catalytic domain and by dot blot using double-stranded PCR fragments containing 5-fC. Many cells and tissues contain very low endogenous levels of 5-fC that may fall below the detection limits of this antibody. This antibody has been validated for specificity using ELISA and dot blot and shows high specificity for 5-fC.
All Species Expected
Monoclonal antibody is produced by immunizing animals with 5-formyl-2'-deoxycytosine.
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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. DRAQ5 is a registered trademark of Biostatus Limited.
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Product # | Size | Price |
---|---|---|
74178S | 100 µl (10 Dot Blots) | $ 113.0 |