A coprecipitation-based validation methodology for interactions identified using protein microarrays

Abstract

Candidate interactions identified by high-throughput protein microarray screening require rigorous -confirmation. Such validation is time-consuming and labor-intensive using conventional techniques. We describe a medium-throughput validation protocol based on coprecipitation of biotin-labeled -proteins synthesized in vitro using a rabbit reticulocyte lysate-coupled transcription and translation system. As our experimental system is based on screening for serum antibodies, we also present methods on purifying immunoglobulin from serum and quantifying the amount of coprecipitated (immunoprecipitated) target protein on Western blot. This technique provides a sensitive confirmatory test allowing for the rapid elimination of false positives prior to more extensive validation and analysis of target interactions in their native environment.