Use of flow cytometric methods to quantify protein-protein interactions

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.

Figure 1

Schematic of FCPIA approach. Avidin-coated microspheres are labeled with biotinylated RGS proteins. The immobilized RGS proteins are incubated with AlexaFluor-532 (Invitrogen, Carlsbad CA) labeled Gα_o. The ability of small molecules or competing unlabeled RGS proteins to inhibit this interaction can be characterized by their ability to diminish bead associated AlexaFluor-532 fluorescence. The assay can be expanded by labeling distinctly identifiable beads (e.g. Luminex bead regions) with different RGS proteins and performing the experiment in a multiplex format.

Figure 2

Saturation of RGS19 binding to Gα_o in the presence or absence of GDP, aluminum fluoride, and magnesium. Non-specific binding is defined by residual affinity of RGS19 for Gα_o-GDP in the absence of aluminum fluoride (-AMF). As can be observed, the interaction between RGS19 and Gα_o binding is dependent on aluminum fluoride and is of high affinity (Kd ∼ 12nM).

Figure 3

Characterization of the RGS19-GIPC protein-protein interaction. Saturation of RGS19 binding to GIPC is independent of which protein is immobilized on the bead. A) Saturation of biotinylated RGS19 by AlexaFluor-532 labeled GIPC. B) Saturation of biotintylated GIPC by AlexaFluor-532 labeled RGS19. Notice that there is no difference in K_d or Bmax. C) immobilized wild type RGS19 is competed by unlabeled wildtype but not ΔPDZ RGS19 for binding to AlexaFluor-532 labeled GIPC. Deletion of the PDZ domain abolishes the affinity of the wild-type RGS19.

Figure 4

Multiplexed flow cytometry analysis during monoclonal antibody development. A) Multiplexed titer analysis of sera from a representative mouse challenged with RGS19. B) Representative single point multiplexed hybridoma screening on 74 clones derived from the mouse in panel A. Notice the high specificity for the target antigen (RGS19) over MBP or RGS4-MBP fusion protein. This method allows a large number of hybridomas (>200) to be screened in a single day for antigenic specificity.