| Assay Method Information | |
| | Thermal Shift Assay |
| Description: | Binding analysis. Protein-ligand binding was identified with the thermal shift assay which is based on the ligand-induced stabilization of the protein tertiary structure. The thermal stability of the ligand-Akt1 complex was assessed by subjecting the complex to a set temperature gradient and by comparison of the meltion temperature of the Akt1-ligand complex with the melting temperature of the protein alone. Protein unfolding was monitored by the fluorescence readout of an environmentally sensitive fluorescent dye, 1-anilinonaphthalene-8-sulfonic acid (ANS). Protein-ligand mixture containing 15 uM compound, 200 ng/mL full length inactive Akt1, 200 uM ANS in the binding buffer (25 mM Tris-HCl (pH. 7.5), 100 mM NaCl, 10% Glycerol, and 5 mM DTT) was prepared in a PCR plate. The PCR plate was placed into a RT-PCR instrument and a temperature gradient was performed, increasing from 27 to 80 C., at 1 C./30 sec heat rate with a dwell time of 20 sec. |
| Affinity data for this assay | |
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