Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions. Protocol 1: Coexpression of Query Protein on NAPPA Slides.

CSH protocols Pub Date : 2008-12-01 DOI:10.1101/pdb.prot5108
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, interactions with a protein of interest (query protein) are detected either by probing an expressed NAPPA slide with the purified query protein or by coexpressing the query protein on the NAPPA slide at the same time that the target proteins are expressed. This protocol describes the coexpression method, which involves adding the gene for the query protein to the cell-free protein expression mix. The amount of query protein that is transcribed and translated from the corresponding plasmid DNA depends on the amount of plasmid DNA used and the size of the protein of interest, among other factors. If too little query protein is expressed, there may be no detectable binding signal. Excessive amounts of protein expression may generate nonspecific background signals. Because the optimum amount of query plasmid varies with each query protein, it is essential to assess empirically the optimal amount of query protein DNA to add to a coexpression experiment.

利用核酸可编程蛋白阵列(NAPPA)鉴定蛋白-蛋白相互作用。方案1:查询蛋白在NAPPA载玻片上的共表达。
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,通过用纯化的查询蛋白探测表达的NAPPA载玻片或在表达目标蛋白的同时在NAPPA载玻片上共同表达查询蛋白来检测与感兴趣蛋白(查询蛋白)的相互作用。本协议描述了共表达方法,包括将查询蛋白的基因添加到无细胞蛋白表达混合物中。从相应的质粒DNA转录和翻译的查询蛋白的数量取决于所使用的质粒DNA的数量和感兴趣的蛋白质的大小,以及其他因素。如果查询蛋白表达过少,可能没有可检测到的结合信号。过量的蛋白表达可能产生非特异性背景信号。由于每个查询蛋白的最佳查询质粒量不同,因此有必要根据经验评估添加到共表达实验中的查询蛋白DNA的最佳量。
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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