Thermal Shift Assay for Apl-1 Binding to HSP90

Protein thermal shift was detected according to the previous description and the manufacturer’s method using HSP90 recombinant protein (1 μg/μL/reaction) and different concentrations of Apl-1 as ligands [21 (link)]. The thermal shift buffer (5 μL) was added, 8X Sypro Orange fluorescent dye (2.5 μL) was added, and the final volume of 20 μL was attained with distilled H₂O. Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific, Waltham, MA, USA) was used to spot the tested compounds at different concentrations on MicroAmp Optical Adhesive Film (Applied Biosystems, Foster City, CA, USA). The reactants were sealed and mixed. A Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect different concentrations of Apl-1 samples on MicroAmp Optical Adhesive Film (Applied Biosystems, Foster City, CA, USA), and the reactants were sealed and mixed. The plate temperature was heated from 25 to 99 °C at a heating rate of 1 °C/min. The detection was undertaken in the Step One Plus Real-Time PCR instrument (Applied Biosystems, Foster City, CA, USA). Displacement software (version 1.3, Applied Biosystems, Foster City, CA, USA) was used to calculate the melting temperatures (Tm) and to establish the thermal curve. The determination of Apl-1 was carried out at different concentrations in triplicate.