{{'Search' | translate}}
 

Bio-Rad Protein Assay Dye Reagent Concentrate, 450 ml

Bio-Rad蛋白测定染料试剂浓缩物

Company: Bio-Rad Laboratories
Catalog#: 5000006
Bio-protocol()
Company-protocol()
Other protocol()

Preparation of Lipid-Stripped Serum for the Study of Lipid Metabolism in Cell Culture
Author:
Date:
2018-06-05
[Abstract]  Studying lipid metabolism in cultured cells is complicated by the fact that cells are typically cultured in the presence of animal serum, which contains a wide, variable, and undefined variety of lipid species. Lipid metabolism can impact cell physiology, signaling, and proliferation, and the ability to culture cells in the absence of exogenous lipids can reveal the importance of lipid biosynthesis pathways and facilitate the generation of media with defined lipid species. We have adapted a protocol to remove lipids from serum without eliminating its ability to support the proliferation of cells in culture. This method requires di-isopropyl ether and butanol and can be used to generate small batches of lipid-stripped serum in four days. The resulting serum supports proliferation of many ... [摘要]  研究培养细胞中的脂质代谢很复杂,因为细胞通常在动物血清存在的情况下进行培养,动物血清含有广泛的,可变的和不确定的脂质种类。 脂质代谢可以影响细胞生理学,信号传导和增殖,并且在不存在外源脂质的情况下培养细胞的能力可以揭示脂质生物合成途径的重要性并促进具有限定的脂质物种的培养基的生成。 我们已经调整了一个方案,以去除血清中的脂质,而不消除其支持培养物中细胞增殖的能力。 该方法需要二异丙醚和丁醇,并且可以在四天内用于生成小批量的脂质剥离血清。 所得血清支持培养中许多细胞系的增殖,并且可用于比较脂质充足和贫化条件下细胞的代谢。

【背景】脂质是细胞膜的主要成分,其描绘生物区室,并且在信号传导途径和能量储存中发挥重要作用(Baenke等人,2013)。脂质代谢在多种疾病中失调,最近的研究表明破坏脂质生物合成可能是抑制肿瘤生长的一种方法(Svensson和Shaw,2016)。因此,有兴趣研究产生细胞脂质物种的代谢途径。在没有特定代谢组分的培养基中培养细胞可以帮助更好地理解代谢途径如何支持细胞功能。大多数哺乳动物细胞的标准培养基包括动物血清作为蛋白质和生长因子(最常见的是胎牛血清[FBS])的来源。动物血清在脂蛋白复合物中含有多种脂质物质并与血清白蛋白结合。不同批次的动物血清组成不同,在大多数实验中血清中的营养物质水平没有明确定义。细胞能够从合成和外源来源获得脂质(Kamphorst等人,2013; ...

Guanine Nucleotide Exchange Assay Using Fluorescent MANT-GDP
Author:
Date:
2018-04-05
[Abstract]  GTPases are molecular switches that cycle between the inactive GDP-bound state and the active GTP-bound state. GTPases exchange nucleotides either by its intrinsic nucleotide exchange or by interaction with guanine nucleotide exchange factors (GEFs). Monitoring the nucleotide exchange in vitro, together with reconstitution of direct interactions with regulatory proteins, provides key insights into how a GTPase is activated. In this protocol, we describe core methods to monitor nucleotide exchange using fluorescent N-Methylanthraniloyl (MANT)-guanine nucleotide. [摘要]  GTP酶是分子开关,在无效GDP结合状态和活性GTP结合状态之间循环。 GTP酶通过其内在的核苷酸交换或通过与鸟嘌呤核苷酸交换因子(GEF)的相互作用来交换核苷酸。 监测体外核苷酸交换,以及与调节蛋白直接相互作用的重构,为GTP酶如何被激活提供了重要见解。 在该协议中,我们描述了使用荧光N-甲基呋喃酰基(MANT) - 鸟嘌呤核苷酸来监测核苷酸交换的核心方法。

【背景】GTPase是鸟嘌呤核苷酸结合蛋白,调节细胞过程的广度,从蛋白质生物合成到细胞周期进展,从细胞骨架重组到膜运输。 GTPases可以被认为是分子开关,它在GDP结合“关闭”状态和GTP结合“开启”状态之间循环;在通过GTP的GDP核苷酸交换结合GTP时,GTP酶变得活跃并且将结合下游效应蛋白以招募和激活这些效应子的生物学功能。 GTP酶通过与开关I环(G2结构域)的高度保守苏氨酸和开关II环(G3结构域)的DxxG基序内的甘氨酸的相互作用结合GTP的γ-磷酸。 GTP水解后,与γ-磷酸相互作用的丧失导致动态构象变化,从而使GTPase变为关闭状态(Vetter and ...

RNA Cap Methyltransferase Activity Assay
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。 ...

Comments