Detection of antimicrobial (poly)peptides with acid urea polyacrylamide gel electrophoresis followed by Western immunoblot.
Detection of antimicrobial (poly)peptides with acid urea polyacrylamide gel electrophoresis followed by Western immunoblot.
复制标题
使用酸性尿素聚丙烯酰胺凝胶电泳检测抗菌（多）肽，然后进行蛋白质免疫印迹。
DOI：
10.1007/978-1-4939-1625-2_7
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Salzman,NitaH
中科院分区：
文献类型：
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作者：
Porter,Edith;Valore,ErikaV;Anouseyan,Rabin;Salzman,NitaH
抗微生物（多）肽（AMP）是先天宿主防御的古老的关键效应分子，并且已经在哺乳动物、昆虫、植物和甚至真菌中鉴定（Nakatsuji和Gallo，J Invest Dermatol，132：887-895，2012）。它们在生理pH下表现出阳离子净电荷并且富含疏水性氨基酸（Dufourc等人，Curr Protein Pept Sci，13：620-631，2012）。它们的作用方式在细菌中得到了最好的研究。当呈现二级结构时，阳离子和疏水性氨基酸被隔离，产生双分区的分子，其中阳离子氨基酸介导与带负电荷的细菌表面的初始静电相互作用，疏水性氨基酸介导嵌入细菌膜中，随后产生干扰细菌活力的多种作用（Nicolas，FEBS J，276：6483-6496，2009; Padovan等人，Curr Protein Pept Sci，11：210-219，2010）。然而，免疫调节、抗肿瘤和其他作用已经被添加到AMP功能的不断增加的列表中（Pushpanathan等人，Int J Pept，2013：675391，2013）。已经基于结构区分了几类AMP，即反平行β-折叠、α-螺旋、环状以及二硫桥连接（Bond和Khalid，Protein Pept Lett，17：1313-1327，2010）。许多AMP经历翻译后修饰，包括进一步的蛋白水解。蛋白质水平的生化分析对许多科学家来说都很有意义，在研究宿主-病原体相互作用时也很重要，例如沙门氏菌入侵小肠。酸性-尿素聚丙烯酰胺凝胶电泳（AU-PAGE），然后进行蛋白质免疫印迹是一个重要的工具，阳离子AMP的鉴定和定量。本文概述的这些程序的方案详细描述了必要的步骤;包括倾倒AU凝胶、制备供试品、进行电泳分离和蛋白转移至膜上，以及使用碱性磷酸酶/NBT/BCIP系统进行免疫检测。标准SDS-PAGE与AU-PAGE的比较和相应的蛋白质免疫印迹如图1所示。
Antimicrobial (poly)peptides (AMPs) are ancient key effector molecules of innate host defense and have been identified in mammals, insects, plants, and even fungi (Nakatsuji and Gallo, J Invest Dermatol, 132: 887–895, 2012). They exhibit a cationic net charge at physiological pH and are rich in hydrophobic amino acids (Dufourc et al., Curr Protein Pept Sci, 13: 620–631, 2012). Their mode of action has been best investigated in bacteria. When assuming secondary structure the cationic and hydrophobic amino acids are sequestered creating a bipartitioned molecule in which the cationic amino acids mediate initial electrostatic interaction with the negatively charged bacterial surface and the hydrophobic amino acids mediate embedding into the bacterial membranes followed by a multitude of effects interfering with bacterial viability (Nicolas, FEBS J, 276: 6483–6496, 2009; Padovan et al., Curr Protein Pept Sci, 11: 210–219, 2010). However, immunomodulatory, antitumor, and other effects have been added to the ever increasing list of AMP functions (Pushpanathan et al., Int J Pept, 2013: 675391, 2013). Several classes of AMPs have been distinguished based on structure, namely anti-parallel beta-sheet, alpha-helical, circular, as well as disulfide bridge connectivity (Bond and Khalid, Protein Pept Lett, 17: 1313–1327, 2010). Many of the AMPs undergo posttranslational modification including further proteolysis. Biochemical analysis at the protein level is of great interest for a wide range of scientists and important when studying host–pathogen interaction, for example Salmonella invasion of the small intestine. Acid-urea polyacrylamide gel electrophoresis (AU-PAGE) followed by Western immunoblotting is an important tool for the identification and quantification of cationic AMPs. The protocol for these procedures outlined here describes, in detail, the necessary steps; including pouring the AU-gels, preparing the test samples, performing the electrophoretic separation and protein transfer to the membrane, and conducting the immunodetection using an alkaline phosphatase/NBT/BCIP system. A standard SDS-PAGE in comparison with AU-PAGE and the corresponding Western immunoblot are depicted in Fig. 1.