Author: Chu, Ruiyin; Reczek, David; Brondyk, William
Title: Capture-stabilize approach for membrane protein SPR assays Document date: 2014_12_8
ID: ueofl0wn_4
Snippet: Whole receptor-based binding kinetics assay. In order to develop GPCR whole receptor assays, a tandem 6xHis/HPC4 tag was added to the C-terminus of full-length human CXCR5 coding sequence and expressed in insect Sf9 cells using the baculovirus expression vector system. Following the methods described by Jaakola V.-P. et al. 16 , the human CXCR5 protein was purified as a mixture of monomer, dimer and trimer (Figure 3a , 3b). The purified receptor .....
Document: Whole receptor-based binding kinetics assay. In order to develop GPCR whole receptor assays, a tandem 6xHis/HPC4 tag was added to the C-terminus of full-length human CXCR5 coding sequence and expressed in insect Sf9 cells using the baculovirus expression vector system. Following the methods described by Jaakola V.-P. et al. 16 , the human CXCR5 protein was purified as a mixture of monomer, dimer and trimer (Figure 3a , 3b). The purified receptor was directly captured on an NTA SPR sensor chip via Ni-mediated affinity capturing. In order to generate a stable chip surface, the commonly used amine coupling reagent NHS/EDC at 20 mM and 5 mM, respectively, was used to stabilize the captured receptor. The surface was then tested for both ligand binding ( Figure 3c ) and antibody binding (Figure 3d ). High quality kinetics data were obtained following injection of ligand or antibody at different concentrations over the receptor surface.
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