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RNA Clean & Concentrator

公司名称: ZYMO RESEARCH
产品编号: R1016
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RNA Cap Methyltransferase Activity Assay
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Date:
2018-03-20
[Abstract]  Methyltransferases that methylate the guanine-N7 position of the mRNA 5’ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [32P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. ... [摘要]  甲基化mRNA 5'帽结构的鸟嘌呤-N7位置的甲基转移酶在真核生物中普遍存在并且通常由病毒编码。这里我们提供生物样品的RNA帽甲基转移酶活性的生化分析的详细方案。该测定包括将含有帽 - 甲基转移酶的样品与[32 P] G-加帽的RNA底物和S-腺苷甲硫氨酸(SAM)温育以产生具有N7-甲基化帽的RNA。然后通过P1核酸酶消化,薄层色谱(TLC)和磷成像确定帽甲基化的程度。此处描述的方案包括用于产生[32 P] G-加帽的RNA底物和用于从哺乳动物细胞制备核和细胞质提取物的附加步骤。该分析也适用于分析其他生物样品(包括重组蛋白制剂和来自分析分离和免疫沉淀/下拉实验的级分)的帽甲基转移酶活性。

【背景】mRNA的5'端的N7-甲基鸟苷帽是适当的真核mRNA加工,定位和翻译所必需的修饰。 ...

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