Epigenase m6A Methylase Activity/Inhibition Assay Kit (Colorimetric)

The Epigenase™ m6A Methylase Activity/Inhibition Assay Kit (Colorimetric) is a complete set of optimized buffers and reagents designed for measuring the activity/inhibition of total m6A methylases (methyltransferases) using nuclear extracts or purified m6A methylases like METTL3/METTL14 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:

• Fast: Colorimetric assay with easy-to-follow steps for convenience and speed. The entire procedure can be finished within 4 hours.
• Robust: Innovative kit composition enables background signals to be extremely low and allows the assay to be simple, accurate, reliable, and consistent.
• Convenient: Both cell/tissue nuclear extracts and purified proteins can be used, which allows detection of inhibitory effects of m6A methylase inhibitor in vivo and in vitro.
• Sensitive and Specific: Novel assay principle allows to detect the m6A methylase-converted end product, which makes the assay much more specific than that for by-product measurement. The assay also 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.
• Quantitative: The assay standard is included, which allows the specific activity of m6A methylase to be quantified. 
• Flexible: 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 and also found in prokaryotes and virus. 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 catalyzed by the methyltransferases (writers) such as METTL3/14, WTAP, RBM15/15B and KIAA1429, and removed by the α-ketoglutarate (α-KG)- and Fe2+-dependent demethylases (erasers) such as FTO, ALKBH5 and TET-like enzymes. It was shown that m6A methylases and demethylases play important roles in many biological processes, ranging from development and metabolism to fertility and participate in pathogenesis of multiple diseases including cancers and viral infections. The dynamic and reversible chemical m6A modification on DNA/RNA may also serve as a novel epigenetic marker of profound biological significance. Up-regulation of m6A modification was shown to increase virus replication and promote cancer growth. Down-regulation of m6A modification was first characterized in human cancer cells and tissues, relative to their normal controls. 

Principle & Procedure
This kit is designed for measuring total m6A methylase activity/inhibition. In an assay with this kit, the unique m6A substrate is stably coated on the strip wells. Active m6A methylases bind to and methylate 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 RNA methylases 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.