Peaq itc instrument

ITC-binding curves were measured by using a Microcal PEAQ-ITC instrument (Malvern). Purified proteins were transferred to buffer containing 20 mM Hepes pH 7.5, 100 mM NaCl, and 2 mM β-mercaptoethanol by 5 mL desalting column (GE). Titrations were performed at 20 °C. Titration of GmPUB13(252-630) in the cell was performed by sequential addition of Avr1d, GmE2, and Avr1d^F90A separately. Data were analyzed using Origin 7.0.

ITC binding experiments were performed on a Microcal PEAQ-ITC instrument with a 19-injection setting. The running parameters were set to: temperature at 25°C, reference power at 5 μcal/s, stir speed at 0.75 rcf/s and the duration/spacing time of 4/150 s. The synthesized peptides were lyophilized and dissolved in the same buffer as MBP-tagged BP1 proteins with 100 mM NaCl, 20 mM HEPES, pH 7.5 and 3 mM β-mercaptoethanol. All the raw ITC data were processed using the Origin 7.0 program.
BLI experiments were carried out using a FortéBio Octet RED 96 system (Pall-fortébio Corporation). The biotinylated histone peptides were loaded onto streptavidin sensors (SA, #18–5019, Pall-fortébio Corporation) in 250 μl of SD buffer (0.1% BSA, 0.05% Tween 20, 10 mM PBS, pH 7.4) for equilibrium for 300 s. The associations of peptide with BP1-6 × His were tested by soaking the peptide-loaded sensors in the wells containing BP1-6 × His at different concentrations for 100 s. The dissociations of peptide from protein were tested by depriving sensors from the wells for 150 s. When the process of association and dissociation for one concentration were finished, 300 s of equilibration was performed to prepare for the next concentration. Finally, the results were calculated by FortéBio data analysis 10.0 software.