Use of flow cytometric methods to quantify protein-protein interactions.
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.
Blazer
Levi L
LL
Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Roman
David L
DL
Muxlow
Molly R
MR
Neubig
Richard R
RR
D001681:Biological Assay; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D005434:Flow Cytometry / Q000379:methods*; D057166:High-Throughput Screening Assays; D011485:Protein Binding; D020710:RGS Proteins / Q000378:metabolism*
D020710:RGS Proteins; C097187:regulator of G-protein signalling 19; D001710:Biotin