Protein thermal shift dye

To each well of a MicroAmp Fast Optical 96-Well Reaction Plate (Applied Biosystems, Foster City, CA, USA), 18 μL of anti-MERS-S2P Fab and 2 μL of Protein Thermal Shift Dye (10×, Applied Biosystems, Foster City, CA, USA) were added. As a negative control, PBS was mixed with Protein Thermal Shift Dye. The plate was sealed with MicroAmp Optical Adhesive Film (Applied Biosystems, Foster City, CA, USA) and centrifuged at 142× g for 1 min. The measurement was performed using a real-time PCR instrument. The instrument was set up according to the manufacturer’s instructions. All the experiments were performed at least in triplicate.

DSF experiments in 384-well format followed a protocol previously established at the SGC-UNICAMP and described elsewhere^{15 (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.

Thermal stabilization assays were performed as described^{28 (link), 56 (link)}. Purified VRK1_3-364 and VRK2_14-335 were screened against a library of 378 structurally diverse, cell permeable ATP-competitive kinase inhibitors purchased from Selleckchem (Houston, TX, USA; catalog No. L1200). DSF experiments were performed in a 384-well plate format. Each well contained 25 μL of 1 μM kinase in potassium phosphate buffer and the Protein Thermal Shift dye at the recommended concentration of 1:1000 (Applied Biosystems; the composition of the buffer and the dye solutions are not disclosed). Compounds (10 mM) in DMSO were added to 16 μM final concentration to complete a total assay volume of 25.8 μL (3.1% final DMSO). Plates were sealed using optically clear films and transferred to a QuantStudio 6 qPCR instrument (Applied Biosystems). Fluorescence intensity data were acquired in a temperature gradient from 25 to 95 °C at a constant rate of 0.05 °C/sec 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 3.1% DMSO was used as a reference.

Thermal stability of the SrLDC proteins was determined by measuring melting curves with the Protein thermal shift dye (Applied Biosystems) in a StepOnePlus Real-Time PCR (Thermo Fisher Scientific) according to manufacturer’s instructions. Briefly, 1 μg of protein was mixed with 1x Protein thermal shift dye (Applied Biosystems) in 20 μl and signal changes reflecting protein denaturation were monitored by increasing temperature from 25 to 90°C. Melting temperatures were determined from the first derivative curve.

The protein thermal shift assay was conducted using the StepOnePlus Real-Time PCR System (Applied Biosystems) with Applied Biosystems Protein Thermal Shift Dye. The T_m of the proteins was calculated using the Protein Thermal Shift software v1.4 (Applied Biosystems).