RNA Cap Methyltransferase Activity Assay
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Author:
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. The protocol described here includes additional steps for generating the [32P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is ...
[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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Preparation, Stimulation and Other Uses of Adult Rat Brain Synaptosomes
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Author:
Date:
2017-12-20
[Abstract] In this paper, our protocol for preparation of brain synaptosomes is described. Synaptosomes are a valuable model system for analysis of structural components of the synapse as well as for investigation of synaptic function. Synaptosomal preparations are necessary for understanding molecular changes at synapses where critical post-translational modifications of synaptic proteins may occur. Not only are synaptosomes rich in synaptic proteins, but they can be used for analyzing uptake of neurotransmitters into synaptic vesicles and for analysis of the involvement of neurotransmitter synthesis and release. Synaptosomes can be stimulated with increased calcium influx to release neurotransmitters. Synaptosomal preparations have been used in characterizing calcium dependent phosphorylation and ...
[摘要] 在这篇论文中,我们描述了制备脑突触体的方案。突触体是用于分析突触的结构组分以及用于调查突触功能的有价值的模型系统。突触体制备对于理解可能发生突触蛋白的关键翻译后修饰的突触处的分子变化是必需的。突触小体不仅含有丰富的突触蛋白,还可用于分析神经递质向突触小泡的摄取和神经递质合成和释放的参与分析。可以用增加的钙内流刺激突触体释放神经递质。突触体制剂已被用于表征钙依赖性磷酸化和GABA合成酶GAD65(分子量为65kDa的L-谷氨酸脱羧酶)的活化。通过检查从突触体制剂获得的突触小泡膜上的蛋白质复合物,可以表征GAD65在GABA囊泡释放的偶联合成和囊泡摄取中的作用,这最终导致GABA囊泡释放GABA能神经传递的微调方法。
【背景】突触体制备方法在40多年前在神经科学研究实验室中建立,并且在涉及神经递质释放相关的细胞外钾升高以及对细胞内钙增加的应答方面具有极大的价值。除了阐明神经递质释放的过程之外,突触体制剂已经成为突触囊泡的有价值的来源。关于突触小泡的研究已经被用于表征参与偶联的神经递质合成和囊泡释放的蛋白质组分。突触体制剂作为突触囊泡分离的中间体也是非常有价值的,然后根据位于包含神经递质合成酶的囊泡膜上的蛋白质复合物进行分析。在这方面关键的意义在于包括CSP(胱氨酸 - ...
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Monitoring the Targeting of Cathepsin D to the Lysosome by Metabolic Labeling and Pulse-chase Analysis
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Author:
Date:
2017-11-05
[Abstract] Mannose 6-phosphate receptors function can be studied in living cells by investigating alterations in processing and secretion of their ligand Cathepsin D. The assay described here is well established in the literature and comprises the metabolic labeling of newly synthesized proteins with [35S] methionine-cysteine in HeLa cells to monitor Cathepsin D processing through secretory pathway and secretion using immunoprecipitation, SDS-PAGE and fluorography.
[摘要] 通过研究其配体组织蛋白酶D的加工和分泌的改变,可以在活细胞中研究甘露糖-6-磷酸受体功能。在此描述的测定在文献中已经很好地确定,并且包括新合成的蛋白质的代谢标记,其中[35 [S]甲硫氨酸半胱氨酸,以监测组织蛋白酶D处理,通过分泌途径和分泌使用免疫沉淀,SDS-PAGE和荧光。
【背景】组织蛋白酶d(CATD)是一种溶酶体天冬氨酸蛋白酶由甘露糖-6-磷酸受体(M6PRs)排序在哺乳动物细胞中该传输它从反面高尔基体网络到内涵体/溶酶体(戈什等人,2003 )。将CatD合成为前体蛋白(〜52kDa),其在溶酶体中被切割以产生中间体(〜48kDa)或成熟的溶酶体形式(〜34kDa)。微量的前体蛋白质也从生物合成途径分泌(Benes et al。,2008)。 catD的丰度可以使用几种方法来确定,例如基于免疫荧光的染色(Poole等人,1972),蛋白质印迹,荧光活性测定(Bewley等人, (Hirst等人,2009; Kametaka等人,2007; Tavares等人,2011)或用脉冲追踪分析进行代谢标记(Hirst等人,2009; Kametaka等人, ...
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