ELISA-Based Quantitation of Global 5hmC Levels

Nelly N. Olova*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract / Description of output

5-Hydroxymethylcytosine (5hmC) is an abundant DNA modification in human and mouse brain, as well as in embryonic stem cells, while severely depleted in multiple types of cancer. Assays for 5hmC detection and quantification, both on a locus-specific and global level, are limited in number and often resource-intensive. Immunodetection of 5hmC through antibodies remains a cost-effective and widely accessible approach. This chapter describes an ELISA-based protocol for 5hmC detection and quantification in genomic or in vitro modified DNA. It is based on the passive adsorption of DNA onto a solid polystyrene surface and the specific detection of 5hmC, which generates a measurable chemiluminescent signal, proportional to the amount of immobilized 5hmC. The assay utilizes a standard curve for interpolation of 5hmC percentage and a loading standard for monitoring loading precision.

Original languageEnglish
Title of host publication TET Proteins and DNA Demethylation
Subtitle of host publicationMethods and Protocols
EditorsOzren Bogdanovic, Michiel Vermeulen
Place of PublicationNew York, NY
Number of pages19
ISBN (Electronic)9781071612941
ISBN (Print)9781071612934
Publication statusPublished - 20 May 2021

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords / Materials (for Non-textual outputs)

  • 5-Hydroxymethylcytosine
  • 5hmC quantitation
  • Antibody detection
  • Chemiluminescence
  • DNA modifications
  • HRP conjugate
  • Immunoassay


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