Thermal Shift Assay for CK2α Protein

Thermal shift binding assay (TSA) was performed with a Bio-Rad CFX real-time PCR system (Hercules, CA, USA). CK2α protein was equilibrated in a buffer (pH 7.5, 100 mM Tris-HCl, 100 mM NaCl) with SYPRO Orange dye (Invitrogen) after addition of DMSO or benzamidine. Samples were freshly prepared and dispensed into 384-well PCR plates at a final volume of 10 μL per well. Fluorescence intensity of each well was measured with a temperature gradient range from 25 to 95 °C with a heating rate increase of 1 °C per minute. To determine the effect of benzamidine on melting temperature (T_m) of CK2α protein, a Boltzmann model was used to generate the protein unfolding curves with GraphPad Prism (v10.0) software.