Membrane Protein Fractionation and Analysis through Western Blot in Aedes aegypti Malpighian Tubules.

Britney Picinic, Jean-Paul V Paluzzi
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Abstract

Western blot analysis is a well-known and dependable technique used to quantify protein abundance in a wide variety of samples. A major consideration for running a successful western blot is ensuring that the protein to be analyzed is purified appropriately. For work with membrane-bound proteins, traditional methods of protein processing such as the use of high-frequency sonication and ultracentrifugation to separate proteins from the membrane are being replaced with less time-consuming approaches. The use of a membrane fractionation kit, which involves the separation of membrane proteins from soluble (cytosolic) proteins, is effective in allowing for the quantification and analysis of membrane-bound proteins. In this protocol, we describe use of the membrane fractionation kit to isolate membrane-bound proteins, followed by western blot analysis, to observe protein abundance. The protocol involves methods that require organ (or tissue) collection, followed by protein processing, and a 2-d western blot procedure.

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埃及伊蚊马氏管的膜蛋白分馏和 Western 印迹分析。
Western 印迹分析是一种众所周知的可靠技术,用于量化各种样本中的蛋白质丰度。成功运行 Western 印迹的一个主要考虑因素是确保要分析的蛋白质经过适当纯化。在处理膜结合蛋白时,传统的蛋白处理方法(如使用高频超声和超速离心从膜上分离蛋白)正被耗时较少的方法所取代。使用膜分离试剂盒可将膜蛋白从可溶性(细胞质)蛋白中分离出来,从而有效地对膜结合蛋白进行定量和分析。在本方案中,我们介绍了使用膜分离试剂盒分离膜结合蛋白,然后进行 Western 印迹分析,以观察蛋白丰度。该方案涉及的方法需要先收集器官(或组织),然后处理蛋白质,再进行二维 Western 印迹程序。
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来源期刊
Cold Spring Harbor protocols
Cold Spring Harbor protocols Biochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (all)
CiteScore
3.00
自引率
0.00%
发文量
163
期刊介绍: Cold Spring Harbor Laboratory is renowned for its teaching of biomedical research techniques. For decades, participants in its celebrated, hands-on courses and users of its laboratory manuals have gained access to the most authoritative and reliable methods in molecular and cellular biology. Now that access has moved online. Cold Spring Harbor Protocols is an interdisciplinary journal providing a definitive source of research methods in cell, developmental and molecular biology, genetics, bioinformatics, protein science, computational biology, immunology, neuroscience and imaging. Each monthly issue details multiple essential methods—a mix of cutting-edge and well-established techniques.
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