Biacore analysis with stabilized G-protein-coupled receptors.
Using stabilized forms of β₁ adrenergic and A₂(A) adenosine G-protein-coupled receptors, we applied Biacore to monitor receptor activity and characterize binding constants of small-molecule antagonists spanning more than 20,000-fold in affinity. We also illustrate an improved method for tethering His-tagged receptors on NTA (carboxymethylated dextran preimmobilized with nitrilotriacetic acid) chips to yield stable, high-capacity, high-activity surfaces as well as a novel approach to regenerate receptor binding sites. Based on our success with this approach, we expect that the combination of stabilized receptors with biosensor technology will become a common method for characterizing members of this receptor family.
Center for Biomolecular Interaction Analysis, University of Utah School of Medicine, Salt Lake City, 84132, USA.
Rebecca L
RL
Rich
James
J
Errey
Fiona
F
Marshall
David G
DG
Myszka
D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D003911:Dextrans / Q000737:chemistry; D007202:Indicators and Reagents / Q000737:chemistry; D007700:Kinetics; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000032:analysis*; D020349:Surface Plasmon Resonance / Q000379:methods*
D003911:Dextrans; D007202:Indicators and Reagents; D043562:Receptors, G-Protein-Coupled; D009571:Nitrilotriacetic Acid