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Recent advances in the field of epigenetics have identified 5-hydroxymethylcytosine (5-hmC) as a key factor in the regulation of gene expression, with substantial implications in the study of tissue differentiation, neurological development, and carcinogenesis. Studies of this epigenetic marker are typically confounded by a lack of reliable methodology for differentiation from the highly prevalent 5-methylcytosine in a DNA sample. The 5-hydroxymethylcytosine pAb has been developed in order to robustly distinguish between hydroxymethylated DNA and methylated or unmodified DNA. Specificity of the antibody is enhanced such that crossreactivity with unmodified and methylated templates is suppressed to near-background levels. The antibody has been extensively tested and validated in ELISA and immunoprecipitation-based enrichment assays, and is suitable for use in further applications including immunohistochemical labeling and chromatographic blotting. Overall, the 5-hydroxymethylcytosine pAb has low cross reactivity with cytosine and 5-methylcytosine versus other available antibodies; can be used with a variety of genomic DNA sources; and has high sensitivity to low masses of 5-hydroxymethylcytosine DNA.
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ELISA analysis: The 5-Hydroxymethylcytosine pAb demonstrates high sensitivity to 5-hydroxymethylcytosine in genomic DNA. In a standard ELISA workflow, 100 ng of purified genomic DNA from several murine tissue sources — brain, kidney, liver, and thymus — are coated per well. The 5-Hydroxymethylcytosine pAb and a secondary antibody (anti-rabbit horseradish peroxidase conjugate) are used at 1:1000 dilution in modified TBS and incubated at 37°C for 1 hour. Optical density was measured after 1 hour of ABTS substrate incubation at room temperature. Independent quantitation of 5-hydroxymethylcytosine levels using LC/MS analysis of genomic DNA from the same tissue sources indicates brain at 0.548%, kidney at 0.225%, liver at 0.107 %, and thymus at 0.030%, demonstrating high correlation with the colorimetric ELISA data.
Immunoprecipitation analysis: Hydroxymethylated DNA is efficiently enriched using the 5-Hydroxymethylcytosine pAb. DNA was immunoprecipitated from 1 ng of a mixed non-methylated/methylated/hydroxymethylated (10:1:1) DNA population. This population was comprised of a mixture of non-methylated plasmid DNA, a methylated version of the same plasmid containing a point mutation that introduces a BamHI restriction site, and a hydroxymethylated version of the same plasmid with a KpnI restriction site. After IP, the region of DNA containing the restriction site was amplified by PCR, digested with either BamHI (B) or KpnI (K), and visualized in a 1.4% (w/v) agarose/TAE/EtBr gel. The results indicate high sensitivity of 5-hydroxymethylcytosine pAb for 5-hydroxymethylcytosine DNA with no detectable crossreactivity to 5-methylcytosine DNA.
| Regulatory Status |
RUO – Research Use Only |
|---|
Related Products
5-Hydroxymethylcytosine DNA ELISA kit
ADI-900-225
Highly sensitive, rapid ELISA kit to quantify 5-hydroxymethylcytosine in DNA with results that closely correlate to mass spectrometry analysis.
| Application | Colorimetric detection, ELISA |
|---|---|
| Assay Time | ~3 hours |
| Sensitivity | <0.02% 5-hydroxymethylcytosine DNA per 100ng input DNA (range 0.03-0.55% (100 ng/µl)) |