A miniaturized sandwich immunoassay platform for the detection of protein-protein interactions

[摘要] BackgroundAnalysis of protein-protein interactions (PPIs) is a valuable approach for the characterization of huge networks of protein complexes or proteins of unknown function. Co-immunoprecipitation (coIP) using affinity resins coupled to protein A/G is the most widely used method for PPI detection. However, this traditional large scale resin-based coIP is too laborious and time consuming. To overcome this problem, we developed a miniaturized sandwich immunoassay platform (MSIP) by combining antibody array technology and coIP methods.ResultsBased on anti-FLAG antibody spotted aldehyde slides, MSIP enables simple, rapid and large scale detection of PPIs by fluorescent labeling anti-myc antibody. By analyzing well-known interacting and non-interacting protein pairs, MSIP was demonstrated to be highly accurate and reproducible. Compared to traditional resin-based coIP, MSIP results in higher sensitivity and enhanced throughput, with the additional benefit of digital read-outs. In addition, MSIP was shown to be a highly useful validation platform to confirm PPI candidates that have been identified from yeast two hybrid systems.ConclusionsIn conclusion, MSIP is proved to be a simple, cost-saving and highly efficient technique for the comprehensive study of PPIs.

[发布日期] 2010-10-28

[关键词] HEK293 Cell;Tandem Affinity Purification;Crude Cell Lysate;Multiplex Array;Human Embryonic Kidney HEK293 Cell