m6A去甲基化酶活性/抑制分析试剂盒（比色法） 货号：A-P-9013
Epigenase m6A Demethylase Activity/Inhibition Assay Kit (Colorimetric)
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背景资料：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%)。
产品描述：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。
产品特点：
1·Colorimetric assay with easy-to-follow steps for convenience and speed. The entire procedure can be finished within 5 hours；
2·Innovative kit composition enables background signals to be extremely low and allows the assay to be simple, accurate, reliable, and consistent；
3.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.
4.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；
5.The assay standard is included, which allows the specific activity of m6A demethylase to be quantified；
6.Strip-well microplate format makes the assay flexible for manual or high throughput analysis；
7.Simple, reliable, and consistent assay conditions；
8.Flexible - Strip-well microplate format makes the assay available for manual or high throughput analysis。
数据分析：
| 操作流程 |
标准曲线 |
柱状图分析 |
 |
 |
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| 使用m6A 去甲基化酶活性/抑制分析试剂盒(比色法)对重组蛋白FTO完成的m6A去甲基化酶活性。重组的人FTO酶以不同的浓度点加入。原始的OD数据。OD与FTO酶活性成反比例 |
使用m6A 去甲基化分析标准品来绘制如上的标准曲线 |
从图示1经过转换得到OD值的数据 |
参考文献：
1.Lewinska A et. al. (February 2017). Downregulation of Methyltransferase Dnmt2 Results in Condition-dependent Telomere Shortening and Senescence or Apoptosis in Mouse Fibroblasts. J Cell Physiol.
保存建议：在接收到艾德寄出的试剂盒后请按照说明书建议，使用不同的保存条件或温度来保存试剂盒内组分。
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