Epigenase m6A Demethylase Activity/Inhibition Assay Kit (Colorimetric)

Product Description

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

Background Information

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.

Starting Materials

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.