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Optional: phosphatase inhibitors if phosphorylated proteins will be detected (Phosphatase Inhibitor Cocktail 3)

磷酸酶抑制剂混合物3

Company: Sigma-Aldrich
Catalog#: P0044
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Mouse Adipose Tissue Protein Extraction
Author:
Date:
2020-06-05
[Abstract]  As obesity becomes a global epidemic, the metabolism research field is increasingly focusing on studying the physiological and pathological roles of adipose tissues (AT). However, extracting proteins from AT is challenging due to abundant fat content of intracellular lipid droplets. Several commercial kits for extraction of AT proteins are available, as are protocols (such as the RELi protocol as well as other protein precipitation protocols). The protocols have been introduced to improve the quality and yield of extractions, but these methods either increase the cost or involve multiple steps. Herein, we describe a detailed protocol for mouse AT protein extractions based on our daily laboratory practice. This protocol requires only very common reagents and instruments, and can be ... [摘要]  [摘要 ] 随着肥胖成为全球性流行病,新陈代谢研究领域越来越集中于研究脂肪组织(AT)的生理和病理作用。然而,由于细胞内脂质滴中脂肪含量丰富,从AT中提取蛋白质具有挑战性。可提供用于提取AT蛋白的商业试剂盒,以及方案(例如RELi 该协议已被引入以提高提取的质量和产量,但是这些方法要么增加了成本,要么涉及多个步骤。在此,我们描述了一种基于小鼠AT蛋白提取的详细协议我们的日常实验室操作。该方案仅需非常通用的试剂和仪器即可完成,可在90-120分钟内完成并提供良好的总蛋白质含量回收率。因此,该方案是一种经济诱人,省时且高效的提取方法。来自AT的蛋白质。

[背景 ] 研究脂肪组织(AT)以解决与肥胖症病理生理学相关的问题通常涉及分析AT蛋白质。然而,AT蛋白质样品中的脂质污染严重影响蛋白质定量的准确性,蛋白质印迹图像的质量以及样品处理下游应用对。为了最大限度地减少脂质污染,商业试剂盒已被开发,如分TM 总蛋白提取试剂盒对脂肪组织/脂肪细胞培养S(发明生物技术公司,2017年)。中号矿全面协议的目标是减少脂肪含量,如过量的脂质(的去除RELI )协议(迪亚兹马林等人,2019)和三氯乙酸(TCA)为基础的蛋白质沉淀法(Benbdelkamel 等人,2018)。尽管提高提取质量已经证实利用在上述过程中,这些方法的缺点也很明显:利用可商购的试剂盒 ...

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。 ...

Extraction of Soluble and Insoluble Protein Fractions from Mouse Brains and Spinal Cords
Author:
Date:
2017-08-05
[Abstract]  The current protocol details the preparation of soluble and insoluble protein lysates from mouse brain or spinal cord samples. In detail, tissue homogenization and sequential protein extraction are described. This procedure yields soluble and insoluble protein extracts that can be further processed in down-stream applications like ELISA or Western blotting. [摘要]  目前的方案详述了从小鼠脑脊髓样品中制备可溶性和不溶性蛋白质裂解物。 详细地描述了组织匀浆和顺序蛋白质提取。 该方法产生可溶性和不溶性蛋白质提取物,其可以在下游应用中进一步加工,如ELISA或Western印迹法。
【背景】这种简单且可重现的脑组织蛋白分离方案详述了总蛋白匀浆物初始分离成可溶性和不溶性级分。 它也可以应用于其他组织样品,并产生含有亲水性蛋白质的可溶性级分和由更疏水的蛋白质组成的不溶性级分。 在不含洗涤剂的裂解缓冲液中进行初始均化后,除去含有可溶性蛋白质级分的上清液,并且可以使用十二烷基硫酸钠(SDS)作为洗涤剂进一步提取含有不溶性级分的沉淀物,以确保全细胞裂解(参见图1)。 这种方法可以通过降低样品的复杂性来促进低丰度蛋白质的分析。


图1.描述顺序提取过程的流程图

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