m6A Demethylase Assay Kit (ab233489)
Key features and details
- Assay type: Enzyme activity (quantitative)
- Detection method: Colorimetric
- Platform: Microplate reader
- Sample type: Nuclear Extracts, Purified protein
Overview
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Product name
m6A Demethylase Assay Kit -
Detection method
Colorimetric -
Sample type
Nuclear Extracts, Purified protein -
Assay type
Enzyme activity (quantitative) -
Product overview
m6A Demethylase Assay Kit (ab233489) 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. m6A demethylase activity may be measured in 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.
A unique m6A substrate is stably coated on the strip wells. Active m6A demethylases bind to and demethylate m 6A 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 undemethylated m6A, which is inversely proportional to enzyme activity, can then be colorimetrically quantified through an ELISA-like reaction.
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Notes
N6-methyladenosine (m6A) is the most common and abundant modification on RNA molecules present in eukaryotes. Recently, DNA m6A was 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 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. The dynamic and reversible chemical m6A modification on DNA/RNA may also serve as a novel epigenetic marker of profound biological significance.
m6A is found to be the most regulated DNA modification in cancers. Thus, determination of such enzymes would be important in benefiting cancer diagnostics and developing new target-based cancer therapeutics.
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Platform
Microplate reader
Properties
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Storage instructions
Please refer to protocols. -
Components 1 x 48 tests 1 x 96 tests 10X m6A Substrate 1 x 10µl 1 x 20µl 10X Wash Buffer 1 x 14ml 1 x 28ml 8-Well Assay Strips (With Frame) 1 x 6 units 1 x 12 units Assay Standard, 2 µg/ml 1 x 10µl 1 x 20µl Binding Solution 1 x 5ml 1 x 10ml Capture Antibody, 1000 µg/ml 1 x 5µl 1 x 10µl Co-Factor 1 1 x 30µl 1 x 60µl Co-Factor 2 1 x 30µl 1 x 60µl Co-Factor 3 1 x 30µl 1 x 60µl Demethylase Buffer 1 x 3ml 1 x 6ml Detection Antibody, 400 µg/ml 1 x 6µl 1 x 12µl Developer Solution 1 x 5ml 1 x 10ml Enhancer Solution 1 x 5µl 1 x 10µl Stop Solution 1 x 5ml 1 x 10ml
Images
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m6A demethylase activity achieved by using recombinant FTO with the m6A Demethylase Assay Kit
Recombinant human FTO enzyme was added at different concentrations. Original OD data. OD is inversely proportional to FTO enzyme activity.
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Standard curve generated with the m6A demethylase assay standardStandard curve generated with the m6A demethylase assay standard -
OD data after conversionOD data after conversion from Fig.1.