A quantum dot-labeled ligand-receptor binding assay for G protein-coupled receptors contained in minimally purified membrane nanopatches.
A robust method to directly measure ligand-receptor binding interactions using fluorescence cross-correlation spectroscopy (FCCS) is described. The example receptor systems demonstrated here are the human micro-opioid receptor, a representative G protein-coupled receptor (GPCR), and Streptavidin, but these general protocols can be extended for the analysis of many membrane receptors. We present methods for the preparation of GPCR-containing membrane nanopatches that appear to have the shapes of nanovesicles, labeling of proteins in membrane vesicles, in addition to the coupling of quantum dots (QDs) to peptide ligands. Further, we demonstrate that reliable binding information can be obtained from these partially purified receptors.
Swift
Jody L
JL
Department of Chemistry, McGill University, Montreal, QC, Canada.
Burger
Melanie C
MC
Cramb
David T
DT
D001681:Biological Assay / Q000379:methods*; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D049329:Nanostructures; D036103:Nanotechnology; D045663:Quantum Dots; D011957:Receptors, Opioid / Q000032:analysis / Q000378:metabolism*; D019809:Streptavidin / Q000737:chemistry* / Q000378:metabolism
D005456:Fluorescent Dyes; D011957:Receptors, Opioid; D019809:Streptavidin