Construction of Protein-Based Biosensors Using Ligand-Directed Chemistry for Detecting Analyte Binding.
Protein-based fluorescent biosensors are powerful tools for quantitative detection of biomolecules or drugs with high sensitivity under physiological conditions. However, conventional methods for construction of biosensors require structural data with high resolution or amino acid sequence information in most cases, which hampers applicability of this method to structurally complicated receptor proteins. To sidestep such limitations, we recently developed a new method that employs ligand-directed chemistry coupled with a bimolecular fluorescence quenching and recovery system, which enabled the conversion of various kinds of membrane-bound receptors to "turn-on" type fluorescent sensors. Here, we describe a protocol for construction of "turn-on" type fluorescent biosensors based on the GABAA receptor which permits quantitative analysis of the ligand affinity.
['Yamaura|Kei|K|', 'Kiyonaka|Shigeki|S|', 'Hamachi|Itaru|I|']
D000818:Animals D015374:Biosensing Techniques D004353:Drug Evaluation, Preclinical D005456:Fluorescent Dyes D058785:GABA-A Receptor Agonists D058787:GABA-A Receptor Antagonists D057809:HEK293 Cells D006801:Humans D008024:Ligands D008856:Microscopy, Fluorescence D008958:Models, Molecular D061848:Optical Imaging D011963:Receptors, GABA-A D013050:Spectrometry, Fluorescence
Fluorescent biosensor Chemical labeling Drug screening GABA(A) receptor Ligand binding Membrane protein