title: Imaging beads-retained prey assay for rapid and quantitative protein-protein interaction.
abstract: 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.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhou
authors.forename: Wanjin
authors.initials: W
authors.lastname: Hong
authors.forename: Lei
authors.initials: L
authors.lastname: Lu
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D045683:Furin / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D016366:Open Reading Frames; D025941:Protein Interaction Mapping / Q000379:methods*; D051381:Rats; D015203:Reproducibility of Results
chemical_list: D055767:Immobilized Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D045683:Furin; D005978:Glutathione
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