Solubilised Lumi4-Tb labelled β2AR was incubated with 10μM BODIPY™ FL L-Cystine dye (Molecular Probes, U.S) with or without 200nM F-propranolol or 100μM cyanopindolol, for 15 mins on ice in 20mM HEPES, 150mM NaCl, 5% glycerol, 0.5% BSA, pH8. For DDM samples 0.1% DDM was used. 20μL samples were added to each well of a 96-well PCR plate and incubated for 30 min over a temperature gradient of 20-78°C across the plate using alpha cycler 2 PCR machine (PCRmax, U.K). Samples were transferred to a 384-well proxiplate (PerkinElmer, U.S). TR-FRET between BODIPY™ FL L-Cystine dye and Lumi4-Tb was read by exciting at 337nm and reading emission at 620nm and 520nm using Pherstar FSX and 337 520/620 module (BMG Labtech). F-propranolol and fluorescent XAC (F-XAC) (CellAura, UK) binding was measured using HTRF 337 665/620 module as above.
Bodipy fl l cystine dye
Manufactured by BMG Labtech
BODIPY™ FL L-Cystine dye is a fluorescent labeling agent used for detecting and quantifying cystine in biological samples. It functions by forming a covalent bond with the sulfhydryl group of cystine, enabling its visualization and analysis.
Automatically generated - may contain errors
2 protocols using bodipy fl l cystine dye
1
Fluorescent Ligand Binding Assay for β2-Adrenergic Receptor
2
Fluorescent Ligand Binding Assay for β2AR
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Solubilised Lumi4-Tb labelled β2AR was incubated with 10μM BODIPY™ FL L-Cystine dye (Molecular Probes, U.S) with or without 200nM F-propranolol or 100μM cyanopindolol, for 15 mins on ice in 20mM HEPES, 150mM NaCl, 5% glycerol, 0.5% BSA, pH8. For DDM samples 0.1% DDM was used. 20μL samples were added to each well of a 96-well PCR plate and incubated for 30 min over a temperature gradient of 20-78°C across the plate using alpha cycler 2 PCR machine (PCRmax, U.K). Samples were transferred to a 384-well proxiplate (PerkinElmer, U.S). TR-FRET between BODIPY™ FL L-Cystine dye and Lumi4-Tb was read by exciting at 337nm and reading emission at 620nm and 520nm using Pherstar FSX and 337 520/620 module (BMG Labtech). F-propranolol and fluorescent XAC (F-XAC) (CellAura, UK) binding was measured using HTRF 337 665/620 module as above.
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