Use of Flow Cytometric Methods to Quantify Protein‐Protein Interactions

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Abstract
Table of Contents
Materials
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Literature Cited

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

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Introduction
Characterizing Protein‐Protein Binding Using the Flow Cytometry Protein‐Protein Interaction Assay
Basic Protocol 1: Saturation Analysis of Biotin‐RGS with Alexa Fluor532‐Gαo
Basic Protocol 2: FCPIA Competition Assay
Basic Protocol 3: High‐Throughput Screening with FCPIA
Support Protocol 1: Biotinylation of RGS Protein
Support Protocol 2: Alexa Fluor532 Labeling of Gαo
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Saturation Analysis of Biotin‐RGS with Alexa Fluor532‐Gαo

Materials

LumAvidin polystyrene beads (L100‐Lxxx‐01, where the “xxx” represents the bead region)
Bead coupling buffer (BCB; see recipe )
Biotinylated RGS protein (see protocol 4 )
Flow buffer (FB; see recipe )
Alexa Fluor532‐conjugated Gα_o (see protocol 5 )
50 µM aluminum chloride, aqueous
50 mM magnesium chloride, aqueous
50 mM sodium fluoride, aqueous

1.5‐ml microcentrifuge tubes
Microcentrifuge, preferably fixed‐angle rotor
Vortex
Full‐skirted 96‐well polypropylene PCR plate (ISC Bioexpress, cat. no. T‐3082‐1)
Multichannel pipet
Aluminum foil
Luminex 200 flow cytometer (Luminex)

Basic Protocol 2: FCPIA Competition Assay

Materials

LumAvidin polystyrene beads (L100‐Lxxx‐01, where the “xxx” represents the bead region)
Bead coupling buffer (BCB; see recipe )
Biotinylated RGS protein (see protocol 4 )
Flow buffer (FB; see recipe )
Compound or competing protein (e.g., unlabeled RGS protein)
Alexa Fluor532‐conjugated Gα_o (see protocol 5 )
50 µM aluminum chloride, aqueous
50 mM magnesium chloride, aqueous
50 mM sodium fluoride, aqueous

1.5‐ml microcentrifuge tubes
Microcentrifuge, preferably fixed‐angle rotor
Vortex
Multichannel pipette or Multidrop micro reagent dispenser (Thermo‐Fisher)
Luminex 200 flow cytometer (Luminex)

Basic Protocol 3: High‐Throughput Screening with FCPIA

Materials

SPHERO streptavidin‐coated particles, 5‐ to 5.9‐µm diameter (Spherotech, SVP‐50‐5)
Bead coupling buffer (BCB; see recipe )
Biotinylated RGS protein (see protocol 4 )
Flow buffer (FB; see recipe )
Test or library compounds
Alexa Fluor‐532‐conjugated Gα_o (see protocol 5 )
50 µM aluminum chloride, aqueous
50 mM magnesium chloride, aqueous
50 mM sodium fluoride, aqueous

1.5‐ml microcentrifuge tubes
Microcentrifuge, preferably with a fixed‐angle rotor
Vortex mixer
Multidrop micro reagent dispenser (Thermo‐Fisher)
Low volume, nonstick, black 384‐well plate (Corning, cat. no. 3676)
HyperCyt liquid sampling unit (IntelliCyt)
Accuri C6 cytometer (Accuri Cytometers)
HyperView software

Support Protocol 1: Biotinylation of RGS Protein

Materials

1 to 10 mg of purified protein, preferably >90% homogeneous
Reaction buffer: 50 mM HEPES, pH 9.0, at 4°C, 250 mM NaCl, 5% glycerol supplemented with 1 mM tris(2‐carboxyethyl)phosphine (TCEP)
N ‐hydroxysuccidimyl ester‐biotin (NHS‐biotin; Sigma‐Aldrich, cat. no. B2643)
Dimethyl sulfoxide (DMSO)
1 M glycine (MP Biomedicals, cat. no. 808831) in 1 M HEPES, pH 9.0, at 4°C
Storage buffer: 50 mM HEPES, pH 7.4, at 4°C, 250 mM NaCl, 5% glycerol supplemented with 1 mM tris(2‐carboxyethyl)phosphine (TCEP)

10‐ml Sephadex G25 gel filtration column (essentially any standard desalting column can be used)
Amicon Ultra centrifugal concentrators (MWCO, 10,000; Millipore, cat. no. UFC901096)

Support Protocol 2: Alexa Fluor532 Labeling of Gαo

Materials

1 to 10 mg of purified Gα_o , preferably >90% homogeneous
Tris(2‐carboxyethyl)phosphine (TCEP)
Reaction buffer: 50 mM HEPES, pH 7.4, at 4°C, 250 mM NaCl, 5% glycerol, supplemented with 10 µM guanosine diphosphate (GDP)
AlexaFluor532 maleimide, 1 mg (Invitrogen, cat. no. A10255)
Dimethyl sulfoxide (DMSO)
1 M dithiothreitol (powdered DTT), aqueous
Storage buffer: 50 mM HEPES, pH 7.4, at 4°C, 250 mM NaCl, 5% glycerol supplemented with 1 mM TCEP and 10 µM GDP

Amicon ultra centrifugal concentrators (MWCO, 10,000; Millipore, cat. no. UFC901096)
10 ml Sephadex G25 gel filtration column (essentially any standard desalting column can be used)
Spectrophotometer

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Figures

Figure 13.11.1 Schematic of FCPIA approach. Avidin‐coated microspheres are labeled with biotinylated RGS proteins. The immobilized RGS proteins are incubated with Alexa Fluor‐532‐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 Alexa Fluor‐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.

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Figure 13.11.2 Saturation of RGS19 binding to Gα_o in the presence or absence of GDP, aluminum fluoride, and magnesium. Nonspecific 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 ( K _d ∼ 12 nM).

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Figure 13.11.3 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 Alexa Fluor‐532‐labeled GIPC. (B ) Saturation of biotinylated GIPC by Alexa Fluor‐532‐labeled RGS19. Notice that there is no difference in K _d or Bmax. (C ) Immobilized wild‐type RGS19 is competed by unlabeled wild‐type but not ΔPDZ RGS19 (dc11RGS19) for binding to Alexa Fluor‐532‐labeled GIPC. Deletion of the PDZ domain abolishes the affinity of the wild‐type RGS19.

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Figure 13.11.4 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.

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Literature Cited

Literature Cited
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Use of Flow Cytometric Methods to Quantify Protein‐Protein Interactions

Abstract

Table of Contents

Materials

Basic Protocol 1: Saturation Analysis of Biotin‐RGS with Alexa Fluor532‐Gαo

Basic Protocol 2: FCPIA Competition Assay

Basic Protocol 3: High‐Throughput Screening with FCPIA

Support Protocol 1: Biotinylation of RGS Protein

Support Protocol 2: Alexa Fluor532 Labeling of Gαo

Figures

Videos

Literature Cited