The Epigenase™ m6A Demethylase Activity/Inhibition Assay Kit (Colorimetric) is a complete set of optimized buffers and reagents to colorimetrically measure the activity/inhibition of total m6A demethylases using nuclear extracts or purified m6A demethylases like FTO and ALKBH5 from a broad range of species such as mammalian, plant, fungal, and bacterial, in a variety of forms including, but not limited to, cultured cells and, fresh and frozen tissues. This kit has the following advantages and features:
- Colorimetric assay with easy-to-follow steps for convenience and speed. The entire procedure can be finished within 5 hours.
- Innovative kit composition enables background signals to be extremely low and allows the assay to be simple, accurate, reliable, and consistent.
- Both cell/tissue nuclear extracts and purified proteins can be used, which allows detection of inhibitory effects of m6A demethylase inhibitor in vivo and in vitro.
- Novel assay principle allows high sensitivity to be achieved. The activity can be detected from as low as 2 µg of nuclear extracts or 50 ng of purified enzymes.
- The assay standard is included, which allows the specific activity of m6A demethylase to be quantified.
- Strip-well microplate format makes the assay flexible for manual or high throughput analysis.
N6-methyladenosine (m6A) is the most common and abundant modification on RNA molecules present in eukaryotes. Recently, DNA m6A is also identified in multicellular eukaryotes including Caenorhabditis elegans and Drosophila melanogaster, and furthermore identified in higher eukaryotes including plants, mouse and human cells. m6A plays crucial roles in regulating DNA replication, DNA damage, RNA splicing, transposition, transcription, and cellular defense. In human cells, the m6A modification is probably catalyzed by a methyltransferase complex METTL3/METTL14 and removed by the α-ketoglutarate (α-KG)- and Fe2+-dependent dioxygenases such as FTO, ALKBH5 and TET-like enzymes. It was shown that METTL3 and α-KG /Fe2+-dependent dioxygenases play important roles in many biological processes, ranging from development and metabolism to fertility. The dynamic and reversible chemical m6A modification on DNA /RNA may also serve as a novel epigenetic marker of profound biological significance. Down-regulation of m6A modification was first characterized in human cancer cells and tissues, relative to their normal controls. m6A is found to be the most regulated DNA modification in cancers. In addition to the regulation in cancer cells, relative to the primary cell/tissues which contain quite low DNA m6A (<0.001%), a hundreds-fold increase of m6A modification was found for in vitro cultured human cells (0.03%-0.22%).
Principle & Procedure
This kit is designed for measuring total m6A demethylase activity/inhibition. In an assay with this kit, the unique m6A substrate is stably coated on the strip wells. Active m6A demethylases bind to and demethylate m6A contained in the substrate. The un-demethylated m6A in the substrate can be recognized with a high affinity m6A antibody and the immuno-signal is enhanced with enhancer solution. The ratio or amount of un-demethylated m6A, which is inversely proportional to enzyme activity, can then be colorimetrically quantified through an ELISA-like reaction.
Input materials can be nuclear extracts or purified enzymes. The amount of nuclear extracts for each assay can be 2 µg to 20 µg with an optimal range of 5 µg to 10 µg. The amount of purified m6A DNA demethylases can be 20 ng to 1 µg with an optimal range of 50 ng to 500 ng, depending on the purity and catalytic activity of the enzymes.