Imaging beads-retained prey assay for rapid and quantitative protein-protein interaction

• Main Authors: Zhou, Y., Hong, Wanjin, Lu, Lei
• Other Authors: School of Biological Sciences
• Format: Article
• Language: English
• Published: 2013
• Subjects: DRNTU::Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/80893; http://hdl.handle.net/10220/9876
• Institution: Nanyang Technological University

Conventional Western blot based pull-down methods involve lengthy and laborious work and the results are generally not quantitative. Here, we report the imaging beads-retained prey (IBRP) assay that is rapid and quantitative in studying protein-protein interactions. In this assay, the bait is immobilized onto beads and the prey is fused with a fluorescence protein. The assay takes advantage of the fluorescence of prey and directly quantifies the amount of prey binding to the immobilized bait under a microscope. We validated the assay using previously well studied interactions and found that the amount of prey retained on beads could have a relative linear relationship to both the inputs of bait and prey. IBRP assay provides a universal, fast, quantitative and economical method to study protein interactions and it could be developed to a medium- or high-throughput compatible method. With the availability of fluorescence tagged whole genome ORFs in several organisms, we predict IBRP assay should have wide applications.