ELISA Kits & Reagents

<h2>What are 5-hmC ELISA kits?</h2>
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5-hmC ELISA kits are antibody-based assays formulated specifically to quantify 5-hydroxymethylcytosine, the oxidized derivative of 5-methylcytosine produced by TET family enzymes, in a genomic DNA sample. The assay typically uses denatured single-stranded DNA coated on a plate well, recognized by an anti-5-hmC primary antibody and developed with an HRP-conjugated secondary antibody for a colorimetric readout proportional to the global level of 5-hmC in the sample.
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<h2 dir="ltr">What you will find:</h2>
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<ul>
<li dir="ltr" role="presentation">DNA hydroxymethylation ELISA assay kits for 5-hmC levels quantification</li>
<li dir="ltr" role="presentation">DNA methylation ELISA kits for comparative gene regulation analysis and measurement of 5-mC&nbsp;</li>
<li dir="ltr" role="presentation">DNA standards for accurate methylation and hydroxymethylation quantification&nbsp;</li>
<li dir="ltr" role="presentation">Control DNA sets and assay controls for experimental accuracy, validation, and reproducibility</li>
<li dir="ltr" role="presentation">HRP-based antibodies for the specific detection in ELISA workflows</li>
<li dir="ltr" role="presentation">ELISA coating and reaction buffers for enhanced assay performance and setup</li>
<li dir="ltr" role="presentation">Signal development reagents for quantitative readout and chemiluminescent or colorimetric detection</li>
</ul>
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<h2>How to use 5-hmC ELISA kits</h2>
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Confirm 5-hmC is detectable at relevant levels in your cell type or tissue
5-hmC is most abundant in neurons and embryonic stem cells, where it can constitute up to 20% of total modified cytosines in neuronal DNA, while most cultured cell lines have much lower 5-hmC levels that may fall below a plate-based antibody assay's reliable detection limit. Checking published 5-hmC abundance for your specific cell type helps confirm whether ELISA-level detection is appropriate before committing samples to the assay.
Use a set of mixed methylation standards to build a calibration curve
Running defined mixtures of 5-hmC-containing and unmethylated DNA at known ratios generates a standard curve that lets you convert the raw absorbance signal to an actual global hydroxymethylation percentage, since the assay signal alone cannot be interpreted as an absolute level without this calibration.
Confirm your antibody's specificity for 5-hmC versus 5-mC
Anti-5-hmC antibodies used in these kits are selected for specificity to 5-hydroxymethylcytosine rather than 5-methylcytosine, but published cross-reactivity data for the specific antibody in your kit is worth checking, particularly when studying sample types where both marks are present at similar levels.
Plan your experimental design to include appropriate negative and positive controls
Including a negative control (unmethylated DNA, expected to give background-level signal) and a positive control (5-hmC-containing DNA of known level) in every plate confirms the assay is performing as expected and catches plate-to-plate variability before it affects result interpretation.
Understand ELISA gives global level, not locus-specific 5-hmC information
A 5-hmC ELISA measures the average hydroxymethylation level across all DNA in the sample; it does not reveal which specific CpG sites carry 5-hmC, which requires hMeDIP-seq or TAB-seq for regional or base-level resolution respectively.
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<h2>Specifications context</h2>
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Anti-5-hmC antibodies used in ELISA formats are validated specifically for 5-hydroxymethylcytosine and selected for minimal cross-reactivity with 5-methylcytosine, since distinguishing these two modifications is the primary purpose of a dedicated 5-hmC assay rather than a general methylation assay. As of 2026, 5-hmC ELISA kits remain a standard first-pass tool in studies where many samples need to be screened for differences in global hydroxymethylation level before committing a smaller subset to high-resolution sequencing methods.
<a href="https://mbpinc.net/contact-us">Contact the expert team</a> at Molecular Biology Products Inc. (MBP) and find reliable ELISA kits and reagents for your lab.

MSDS

Cytosine DNA Standard (2 µg)

D5405-1 by Zymo Research is the Cytosine DNA Standard (2 µg) containing 100% unmodified cytosines (no 5-mC, no 5-hmC), used to anchor the zero-modification point on calibration curves in 5-mC and 5-hmC ELISA quantitation assays. Compatible with Zymo Research 5-mC DNA ELISA Kit (D5325) and the 5-Methylcytosine &amp; 5-Hydroxymethylcytosine DNA Standard Set (D5405). Store at −20 °C. Made in USA.

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HRP Developer

D5425-4-15 by Zymo Research is HRP Developer (15 ml), a colorimetric substrate solution for use with the Quest 5-hmC DNA ELISA Kit and Quest 5-mC DNA ELISA Kit. Sold individually for component replenishment or as part of a complete kit. Enables quantitative measurement of genomic 5-hmC and 5-mC levels. Also available in 30 ml (D5425-4-30). MBP is a registered vendor for Howard Hughes Medical Institute, Vanderbilt University, MD Anderson Cancer Center, Eurofins, and many other institutes across the USA.

HRP Developer (15 ml)

HRP Developer (30 ml)

DataSheet

Protocol

Quest 5-hmC™ DNA ELISA Kit

The Quest 5-hmC™ DNA ELISA Kit (D5425) by Zymo Research quantifies global 5-hydroxymethylcytosine in 20–200 ng of genomic DNA per well with a sensitivity of ≥0.02% 5-hmC per 100 ng input. The 96-well colorimetric assay completes in 3 hours and is compatible with vertebrate, plant, and microbial DNA, as well as enzyme-digested and sheared fragments.