Description
Key facts
Detection method:Colorimetric,
Sample types:Nuclear Extracts, Purified protein,
Assay type:Enzyme activity (quantitative),
Assay Platform:Microplate reader
Product details:
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.
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
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 Fe
-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.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions-Multi, Appropriate long-term storage conditions-Multi, Storage information-Please refer to protocols
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
The m6A demethylase also known as FTO (Fat Mass and Obesity-associated protein) or ALKBH5 is an enzyme involved in the removal of methyl groups from RNA molecules. This protein has a significant molecular mass around 58 kDa depending on post-transcriptional modifications. M6A demethylase is expressed in multiple tissues including the brain heart liver and adipose tissue. The enzyme plays a role in regulating the stability and translation of mRNA by demethylating N6-methyladenosine (m6A) modifications an important reversible epitranscriptomic modification.
Biological function summary
The m6A demethylase impacts the regulation of gene expression by modulating RNA metabolism. It works as part of a complex that involves other m6A-related enzymes such as m6A methyltransferases and m6A binding proteins. The demethylation activity contributes to processes like RNA splicing nuclear export and degradation which influence cell differentiation and development. Its activity is key in controlling mRNA fate and function in response to environmental and cellular signals.
Pathways
The m6A demethylase associates closely with the mTOR signaling pathway and the AMPK signaling pathway. In the mTOR pathway the demethylase regulates nutrient sensing and energy homeostasis by modulating RNA stability impacting proteins like mTOR itself and S6 kinase. In the AMPK pathway the demethylase interacts with key regulators of cellular energy status adjusting the expression of targets involved in metabolic processes and autophagy.
The involvement of m6A demethylase in obesity and cancer has been widely documented. Alterations in FTO a well-known m6A demethylase have links to obesity due to its role in metabolic regulation through the mTOR pathway. In cancer mutations affecting ALKBH5 another demethylase influence tumor progression and treatment resistance owing to altered mRNA metabolism pathways. The interplay between m6A demethylase mTOR and AMPK signaling shows profound impact on both metabolic disorders and oncogenic processes.
