| Assay Method Information | |
| | Measurement of Dissociation Constants (Kd) by SPR |
| Description: | SPR experiments were performed using the P4SPR from Affinité Instruments using Ni-NTA immobilization chips and his-tagged protein. Ni-NTA coated surfaces allow the immobilization of his-tagged proteins by chelation of histidine residues to the nickel ion. The sensor chip was inserted into a quad inlet model P4SPR (with 4 independent channels). Once the instrument was turned on, the baseline was stabilized by deionized (DI) water, followed by signal stabilization by the running buffer. His-tagged protein at 10 μg/mL was injected into all 4 channels of the P4SPR and was left to react for 20 min. The sensor chip was then washed with DI water. The lowest concentration of the ligand was injected into the channels of the P4SPR and was left to react for 10 min. The SPR shift was saved. Then, a higher concentration of the ligand was injected, and the sample injection steps were repeated until all 5 concentrations have been added. The KD of the binding interaction between the ligand and protein was determined by using the affinity curve fitting function in the P4SPR Control software. |
| Affinity data for this assay | |
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