Link Andrew J, Labaer Joshua
Vanderbilt University School of Medicine, Nashville, TN 37232-8575, USA.
CSH Protoc. 2008 Dec 1;2008:pdb.ip62. doi: 10.1101/pdb.ip62.
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.
引言
用于生产蛋白质微阵列的核酸可编程蛋白质阵列（NAPPA）方法利用无细胞提取物将编码靶蛋白的cDNA直接转录并翻译到载玻片上。在制备阵列之后，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。第一种是用感兴趣的纯化蛋白（查询蛋白）探测已表达的NAPPA载玻片并寻找相互作用蛋白。这些相互作用的信号可以通过直接标记查询蛋白或使用针对蛋白本身或蛋白上标签的标记抗体来检测。当能够获得纯化蛋白时，这种方法效果良好，并且具有用户可以在有或没有翻译后修饰的情况下或在各种结合条件下测试查询蛋白结合的优点。第二种模式需要在表达所有靶蛋白的同时在NAPPA载玻片上共表达查询蛋白。本文描述了使用共表达查询蛋白的优点，并提供了选择合适表位标签的建议。
