| Assay Method Information | |
| | Receptor Binding Assay |
| Description: | 1. Experimental Materials1.1 Reagent MaterialsLigand: LANCL2Running buffer: 20 mM MES, 150 mM NaCl, 0.05% P20, pH 6.5, 1% DMSO1.2 EquipmentsEquipment name: Biacore S200Chip type: CM5 (29-1496-03)2. Experimental Methods2.1 Ligand CouplingLANCL2 protein was immobilized, and diluted to 50 μg/mL using sodium acetate solution at pH 4.0.Injection conditions: the CM5 chip surface was activated with a mixture of EDC/NHS with a flow rate of 10 μL/min and an injection time of 420 s; then LANCL2 injection was performed with a flow rate of 5 μL/min and an injection time of 2000 s, and the ligand coupling volume each time was about 1800 RU; finally, the chip surface was blocked with ethanolamine with a flow rate of 10 μL/min and an injection time of 420 s.Coupling buffer: 20 mM MES, 150 mM NaCl, 0.05% P20, pH 6.5.2.2 Experimental ConditionsAnalytes: All sample analytes of small molecule compounds were diluted 2-fold from a concentration of 50 μM to a concentration of 0.78 M, so as to obtain a compound solution containing 1% DMSO.Injection conditions for small molecule compounds: a flow rate of 30 μL/min, a binding time of 60 s, a dissociation time of 300 s.Running buffer: 20 mM MES, 150 mM NaCl, 0.05% P20, pH 6.5, 1% DMSO.Sample chamber temperature: 25° C.; analysis temperature: 25° C.MethodsKinetic determination of LANCL2-small molecule interactions was performed. The kinetic parameters of small molecules BT-11 and L-1 to 60 (analytes) binding to LANCL2 (ligand) were determined using BIACORE S200. Data were generated in triplicate in a dose-dependent manner (5-8 titration points) and analyzed to determine binding models (Langmuir, conformational shifts, etc.), real-time association and dissociation constants, and equilibrium dissociation constants. SPR technology allows the validation of specific LANCL2-phytochemical interactions as well as an increased insight into the gold standard of binding mechanisms and rates. Experiments were performed on carboxymethyl polydextrose (CM5) sensor chips by amine-coupled covalent attachment of LANCL2. Data were analyzed with BIACORE S200T200 evaluation software (version 1), so as to determine affinity binding constants (KD) using a 1:1 binding model. |
| Affinity data for this assay | |
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