| Assay Method Information | |
| | Measurement of Dissociation Constants (K) by NMR |
| Description: | Dissociation constants were obtained by monitoring changes in chemical shifts as a function of ligand concentration. The changes in chemical shifts (d) were calculated according to the following equation (Williamson, Progress in Nuclear Magnetic Resonance Spectroscopy, Volume 73, August 2013, Pages 1-16): d=√(1/2[δ_H{circumflex over ( )}2+(α−δ_N{circumflex over ( )}2)]), Where δ are the changes in chemical shift in ppm for 1H and 15N and the correction factor (α) was set at 0.15.1H 1D protein-observed experiments were recorded with 15-50 μM protein. The standard Bruker 1D 1H sequence with excitation sculpting (zgesgp) was employed. A relaxation delay of 1 s was employed in order to use 256 scans while still keeping experimental time fairly short. Changes in protein chemical shift or peak intensity in the methyl region were monitored against compound concentration. |
| Affinity data for this assay | |
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