Profiling Interactome Networks with the HaloTag-NAPPA In Situ Protein Array

Q1 Agricultural and Biological Sciences

Current protocols in plant biology Pub Date : 2018-08-14 DOI:10.1002/cppb.20071

Junshi Yazaki, Mary Galli, Alice Y. Kim, Joseph R. Ecker

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引用次数: 5

Abstract

Physical interactions between proteins and other molecules can be evaluated at a proteome scale using protein arrays, a type of high-throughput pulldown assay. We developed a modified in situ protein array known as the nucleic acid programmable protein assay (NAPPA) that allows the screening of thousands of open reading frames (ORFs) at a lower cost, with less labor, and in less time than conventional protein arrays. The HaloTag-NAPPA protein array can efficiently capture proteins expressed in situ on a glass slide using the Halo high-affinity capture tag. Here, we describe the fabrication of the array using publicly available resources and detection of protein-protein interactions (PPIs) that can be used to generate a protein interactome map. The Basic Protocol includes procedures for preparing the plasmid DNA spotted on glass slides, in situ protein expression, and PPI detection. The supporting protocols outline the construction of vectors and preparation of ORF clones. © 2018 by John Wiley & Sons, Inc.

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利用HaloTag-NAPPA原位蛋白阵列分析相互作用组网络

蛋白质和其他分子之间的物理相互作用可以使用蛋白质阵列在蛋白质组尺度上进行评估，这是一种高通量下拉试验。我们开发了一种改良的原位蛋白阵列，称为核酸可编程蛋白测定(NAPPA)，与传统的蛋白阵列相比，它可以以更低的成本、更少的劳动力和更短的时间筛选数千个开放阅读框(orf)。HaloTag-NAPPA蛋白阵列可以使用Halo高亲和力捕获标签有效地捕获在玻璃载玻片上原位表达的蛋白。在这里，我们描述了阵列的制造使用公开可用的资源和检测蛋白质-蛋白质相互作用(PPIs)，可用于生成蛋白质相互作用组图。基本方案包括制备在玻片上斑点的质粒DNA、原位蛋白表达和PPI检测的程序。支持协议概述了载体的构建和ORF克隆的制备。©2018 by John Wiley &儿子,Inc。

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Current protocols in plant biology Agricultural and Biological Sciences-Plant Science

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. Current Protocols in Plant Biology provides reproducible step-by-step instructions for protocols relevant to plant research. Furthermore, Current Protocols content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols in Plant Biology to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Plant Biology is the comprehensive source for protocols in the multidisciplinary field of plant biology, providing an extensive range of protocols from basic to cutting edge. Coverage includes: Extraction and analysis of DNA, RNA, proteins Chromosome analysis Transcriptional analysis Protein expression Metabolites Plant enzymology Epigenetics Plant genetic transformation Mutagenesis Arabidopsis, Maize, Poplar, Rice, and Soybean, and more.