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

PLoS One. 2013;8(3):e59727. doi: 10.1371/journal.pone.0059727. Epub 2013 Mar 29.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cloning, Molecular
  • Cytosol / metabolism
  • Furin / chemistry
  • Glutathione / chemistry
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Immobilized Proteins / metabolism
  • Luminescent Proteins / chemistry*
  • Mice
  • Microscopy, Fluorescence / methods*
  • Open Reading Frames
  • Protein Interaction Mapping / methods*
  • Rats
  • Reproducibility of Results

Substances

  • Immobilized Proteins
  • Luminescent Proteins
  • Green Fluorescent Proteins
  • Furin
  • Glutathione

Grant support

This work is supported by the following grants to LL: Nanyang Technological University (SUG), Ministry of Education (RG18/11) and Ministry of Health (NMRC/CBRG/0007/2012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.