About Quest 5-hmC™ DNA ELISA Kit
The 5-methylcytosine (5-mC) modification of DNA in epigenetic regulation has been well studied over the last several decades. However, the role of the so-called “sixth base”, 5-hydroxymethylcytosine (5-hmC), has yet to be defined. Recent studies have associated 5-hmC patterns to transcriptional regulation of genes, which may contribute to normal and disease states of organisms.
The Quest 5-hmC™ DNA ELISA Kit is both sensitive and specific and can be used to accurately detect 5-hmC DNA in a variety of samples. The kit is compatible with a wide range of input DNA including intact vertebrate, plant, and microbial genomic DNA, as well as enzyme-digested and mechanically sheared fragments. The Control DNA Set included with this kit has been calibrated to accurately quantify the percent 5-hmC in sample DNA by use of a standard curve. Also, the fast, streamlined workflow is ideal when analyzing/screening large numbers of samples.
| Equipment | An incubator and plate reader (w/ 405-450 nm detection) are required. A multi-channel pipettor is recommended. An automated plate washer may be used for blocking and wash steps due to the one-buffer system. |
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| Sample Sources | This ELISA procedure has been optimized for the detection of 5-hmC in purified genomic DNA that is intact, sheared or fragmented in PBS, Tris-EDTA, or water. The product is also compatible with DNA from other sources. |
| Detection | This system is highly sensitive for 5-hmC DNA and has a lower detection of 0.02% per 100ng input DNA, making it easy to detect even the smallest percentage of 5-hmC in DNA samples. |
 Quest 5-hmC™ DNA ELISA Kit is a sandwich-based ELISA format. First, Anti-5-Hydroxymethylcytosine Polyclonal Antibody (anti-5-hmC pAb) is coated to the bottom of a well. Single stranded 5-hmC-containing DNA binds to anti-5-hmC pAb which is then recognized by Anti-DNA HRP Antibody. Addition of HRP Developer will produce a greenish-blue color in the wells containing 5-hmC DNA.
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 The Quest 5-hmC™ DNA ELISA Kit can be used to detect 5-hmC in numerous DNA samples with high specificity as evidenced by comparison with LC-MS/MS-MRM analysis. 5-hmC pAb (200 ng/well) was used to quantitate the amount 5-hmC in 100 ng of single-stranded DNA. For this, % 5-hmC was calculated from a standard curve generated using the Control DNA Set. The figure shows a correlation between the % 5-hmC in DNA samples calculated using the Quest 5-hmC™ DNA ELISA Kit (ELISA) and mass spectrometry (MS).
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