Unit

UNIT 13.11 Use of Flow Cytometric Methods to Quantify Protein-Protein Interactions

  1. Levi L. Blazer1,
  2. David L. Roman4,
  3. Molly R. Muxlow1,
  4. Richard R. Neubig1,2,3

Published Online: 1 JAN 2010

DOI: 10.1002/0471142956.cy1311s51

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Blazer, L. L., Roman, D. L., Muxlow, M. R. and Neubig, R. R. 2010. Use of Flow Cytometric Methods to Quantify Protein-Protein Interactions. Current Protocols in Cytometry. 51:13.11:13.11.1–13.11.15.

Author Information

  1. 1

    Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan

  2. 2

    Department of Internal Medicine, The University of Michigan Medical School, Ann Arbor, Michigan

  3. 3

    Center for Chemical Genomics, The University of Michigan Medical School, Ann Arbor, Michigan

  4. 4

    Division of Medicinal and Natural Products Chemistry, The University of Iowa, Iowa City, Iowa

Publication History

  1. Published Online: 1 JAN 2010
  2. Published Print: JAN 2010

Abstract

A method is described for the quantitative analysis of protein-protein interactions using the flow cytometry protein interaction assay (FCPIA). This method is based upon immobilizing protein on a polystyrene bead, incubating these beads with a fluorescently labeled binding partner, and assessing the sample for bead-associated fluorescence in a flow cytometer. This method can be used to calculate protein-protein interaction affinities or to perform competition experiments with unlabeled binding partners or small molecules. Examples described in this protocol highlight the use of this assay in the quantification of the affinity of binding partners of the regulator of G-protein signaling protein, RGS19, in either a saturation or a competition format. An adaptation of this method that is compatible for high-throughput screening is also provided. Curr. Protoc. Cytom. 51:13.11.1-13.11.15. © 2010 by John Wiley & Sons, Inc.

Keywords:

  • RGS;
  • G protein;
  • protein-protein interaction;
  • FCPIA;
  • high-throughput screening;
  • multiplexing