DSF experiments in 384-well format followed a protocol previously established at the SGC-UNICAMP and described elsewhere15 (link). Briefly, CAMKK2-KD protein was screened against a library of 378 structurally diverse ATP-competitive kinase inhibitors available from Selleckchem (Houston, TX, United States; catalog No. L1200). Each well contained 20 μL of 1 μM kinase in 100 mM potassium phosphate pH 7.0, 150 mM NaCl, 10% glycerol and the Applied Biosystems Protein Thermal Shift dye at the recommended concentration of 1:1000.
The compounds, previously solubilized in DMSO, were used at 10 µM final concentration and 0.1% DMSO. Plates were sealed using optically clear films and transferred to a QuantStudio 6 qPCR instrument (Applied Biosystems). The fluorescence intensity was measured during a temperature gradient from 25 to 95 °C at a constant rate of 0.05 °C/s, and protein melting temperatures were calculated based on a Boltzmann function fitting to experimental data, as implemented in the Protein Thermal Shift Software (Applied Biosystems). Protein in 0.1% DMSO was used as a reference.
The compounds, previously solubilized in DMSO, were used at 10 µM final concentration and 0.1% DMSO. Plates were sealed using optically clear films and transferred to a QuantStudio 6 qPCR instrument (Applied Biosystems). The fluorescence intensity was measured during a temperature gradient from 25 to 95 °C at a constant rate of 0.05 °C/s, and protein melting temperatures were calculated based on a Boltzmann function fitting to experimental data, as implemented in the Protein Thermal Shift Software (Applied Biosystems). Protein in 0.1% DMSO was used as a reference.
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