Nanodsc system

The melting temperatures of wild-type and mutant Tm-ACPs were measured by DSC using a NanoDSC system (TA instruments, New Castle, DE, USA). The protein samples were prepared at concentrations of 2 mg/mL in 20 mM potassium phosphate buffer (pH 7.0). After degassing for 10 min, the reference buffer and the protein samples were equilibrated at 50 °C for 10 min. The thermograms were recorded as the temperature was increased at a rate of 1°C/min from 50 °C to 120 °C. During the measurements, the pressure was kept constant at 3 atm to prevent the phase transition of the solvent. After polynomial baseline corrections and two-state scaled curve fittings, individual component peaks were resolved from the complex profiles.

Purified antibodies at a concentration of 1 mg/mL in buffer (20 mM citrate, 100 mM NaCl, pH 6.2) were submitted to the Nano DSC system (TA instrument) for analysis. A temperature ramp of 1°C/min was performed with monitoring from 25 to 100°C. Thermograms of the blank buffer were subtracted from each antibody prior to analysis and the Tm values were calculated after deconvolution using the Nano DSC software.

DSC measurements were performed using a Nano DSC system (TA). Prior to scanning, samples were degassed under vacuum for 15 min using a degassing station (TA). DSC thermograms were determined by monitoring the difference in heat capacity in solution upon increasing temperature at a scan rate of 1 °C min⁻¹ by heating the sample from 15 °C to 75 °C under increased pressure (3 atm). All proteins used in this study were extensively dialyzed against a buffer containing 50 mM NaCl, 50 mM Tris, pH 7.6, and the dialysis buffer was used for instrumental baseline scans and as reference samples. Protein concentrations used were 1.0 mg/ml, corresponding to 75.0 μM for ARID4A/ARID4B TD121 proteins. Data were fitted to a two-state scaled model using NanoAnalyze software.