| Assay Method Information | |
| | SPR Protocol to Measure Binding of a Compound to FKBP12 |
| Description: | This protocol utilizes Surface Plasmon Resonance (SPR) as a method to determine kinetics (KD, Ka, Kd) for the binding of compound (analyte) to immobilized FKBP12 (ligand).Reagents:Compound in 100% DMSO (in-house), 10×HBS-P+ buffer (GE Healthcare BR-1006-71), Assay buffer (1×HBS-P+ buffer, 1% DMSO), 12×HIS tagged FKBP12 (in-house).Equipment:BIACORE X100 (GE Healthcare)Supplies:NTA Sensor chip (GE Healthcare BR-1000-34)Experimental Protocol:Experiments are performed at 25° C. Stock solution of 12×HIS tagged FKBP12 is diluted to 100 nM in assay buffer (1% DMSO final). Approximately 500-600 RU of FKBP12 is immobilized on one of two flow cells of an activated NTA chip. The second flow cell is not activated as a reference for non-specific interaction of the analyte to the sensor chip. Various concentrations of compound (1 nM-1 μM range), serially diluted into the same assay buffer (1% DMSO final), are injected onto the FKBP12 surface and reference surface at a flow rate of 10 μl/min. The surface is regenerated between analyte injections with 350 mM EDTA.Data Fitting:The BiaEvaluation software program is used for data fitting. All data is reference subtracted against both the reference flow cell and a buffer injection. For kinetic analyses, data is locally fit to a 1:1 interaction model. |
| Affinity data for this assay | |
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