Air-stable G protein-coupled receptor microarrays and ligand binding characteristics.
This paper described novel strategies to achieve air-stable G protein-coupled receptor (GPCR) microarrays and the uses of the microarrays for ligand profiling. Specifically, GPCR cell membrane fragments were suspended in a buffered solution containing bovine serum albumin (BSA) and disaccharide sucrose or trehalose and used for fabricating GPCR microarrays. During the array fabrication and postfabrication processes, BSA molecules were found to effectively form packed layer(s) surrounding the GPCR membranes immobilized onto the predetermined printing area, thereby stabilizing the membrane microspots. The use of disaccharides was shown to protect the integrity and functionality of GPCR microarrays from the typical deterioration of the membranes when fabricated and stored under dry conditions. To utilize the ability of fluorescence technology for multichannel detection as well as to maximize the capability of GPCR microarrays for multiplexed binding assays, several fluorescently labeled ligands were synthesized and optimized for multiplexing binding assays. A schematic microarray of five GPCRs had been used as a model for characterizing the association and dissociation rate constants of labeled ligands binding to their respective receptors in the microarrays. Interestingly, distinct receptor-ligand interactions exhibited different dependence on the type of pH reagent as well as the species and concentration of cations used in a binding assay buffered solution. The potential mechanisms and implications for the uses of air-stable GPCR microarrays were discussed.
Fang
Ye
Y
Biochemical Technologies, Science and Technology Division, Corning Incorporated, Sullivan Park, Corning, New York 14831, USA. fangy2@corning.com
Peng
Jinlin
J
Ferrie
Ann M
AM
Burkhalter
Robert S
RS
D000388:Air; D000818:Animals; D002021:Buffers; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000032:analysis* / Q000378:metabolism; D013678:Technology, Pharmaceutical
D002021:Buffers; D008024:Ligands; D043562:Receptors, G-Protein-Coupled