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Assay Method Information |
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| | ITC |
| Description: | All ITC measurements were recorded at 25 °C with an AutoITC200 microcalorimeter (MicroCal Inc.). All protein and compound stock samples were in the target buffer (25 mM Tris-HCl, pH 8, 150 mM NaCl, and 2 mM β-mercaptoethanol), and then diluted in the same buffer to achieve the desired concentrations: 90 200 μM protein and 1 2 mM compound depending on the expected dissociation constant. The concentration of protein stock solutions were established using the Edelhoch method, whereas 10 mM compound stock solutions were prepared gravimetrically based on molecular weight. A typical experiment included a single 0.2 μL compound injection into a 200 μL cell filled with protein, followed by 25 subsequent 1.5 μL injections of compound. Injections were performed with a spacing of 180 seconds and a reference power of 8 μcal/sec. |
| Affinity data for this assay | |
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