Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions: General Guidelines.

CSH protocols Pub Date : 2008-12-01 DOI:10.1101/pdb.ip62
Andrew J Link, Joshua Labaer
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引用次数: 1

Abstract

INTRODUCTIONThe Nucleic Acid Programmable Protein Array (NAPPA) approach for producing protein microarrays uses cell-free extracts to transcribe and translate cDNAs encoding target proteins directly onto glass slides. Following array preparation, the identification of protein interactions using NAPPA can be accomplished by either of two general schemas. The first is to probe an expressed NAPPA slide with a purified protein of interest (the query protein) and look for interactors. Signals of these interactions can be detected either by directly labeling the query protein or by using a labeled antibody either to the protein itself or to a tag on the protein. This approach works well when there is access to the purified protein, and it has the advantage that users can test query protein binding with and without post-translational modifications or under a variety of binding conditions. The second schema entails coexpressing the query protein on the NAPPA slide at the same time that all the target proteins are expressed. This article describes the advantages of using a coexpressed query protein and provides advice on choosing a suitable epitope tag.

使用核酸可编程蛋白阵列(NAPPA)鉴定蛋白质-蛋白质相互作用:一般指南。
核酸可编程蛋白阵列(NAPPA)方法用于生产蛋白质微阵列使用无细胞提取物转录和翻译编码靶蛋白的cdna直接到玻片上。在阵列制备之后,使用NAPPA鉴定蛋白质相互作用可以通过两种一般模式中的任何一种来完成。第一种方法是用感兴趣的纯化蛋白(查询蛋白)探测表达的NAPPA载玻片并寻找相互作用物。这些相互作用的信号可以通过直接标记查询蛋白或使用标记抗体对蛋白质本身或蛋白质上的标签进行检测。当有途径获得纯化蛋白时,这种方法效果很好,并且它的优点是用户可以测试查询蛋白结合是否有翻译后修饰或在各种结合条件下的结合。第二种模式需要在表达所有目标蛋白的同时共同表达NAPPA载玻片上的查询蛋白。本文介绍了使用共表达查询蛋白的优点,并提供了选择合适的表位标签的建议。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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