Measuring ARHGAP18-Ezrin/FERM Binding by ITC

Purified ARHGAP18 was produced as described in the antibody production except that the SUMO tag was not cleaved as it was found that the protein was insoluble above 2 mg/mL without it. Purified ezrin and FERM were produced as described in Viswanatha et al., 2012 (link). Ezrin or FERM were diluted to 75–80 µM depending on the purified volume and number of injections needed for ITC. The proteins were then injected into 30 µM SUMO-ARHGAP18 using automated injections on a TA Instruments-Waters LLC (New Castle, DE) Affinity ITC-LV. Injections were performed using ITC run software (TA Instruments) with a minimum pulse time of 200 s and 2–5 μL injections and a stirring rate of 175 rotations per minute. The system was calibrated using the injection of water into water as a negative control and the injection of 0.95 mM CaCl₂ into 0.150 mM EDTA as a positive control. Injections were continued until saturation of the binding was observed through a plateau of the released energy per injection or the max volume of the injection syringe was depleted. Collected ITC data were then imported into NanoAnalyze software (TA Instruments), and a baseline linear curve representing the energy from injecting water into water was subtracted leaving a normalized energy injection dataset. Injection points from either the first injection or injections after the binding affinity had plateaued were excluded and the remaining data points were then fit to a single independent binding curve. FL-ezrin or FERM injections that did not show an absorbance curve were not fit to one in the NanoAnalyze program as part of a pre-exclusion criteria.