2.9. Biolayer Interferometry

Measurements of the kinetics of Fbp2–Camk2a interaction was performed using ForteBio Octet K2 (Pall ForteBio, Fremont, CA, USA) and high-specificity anti-His antibody biosensor (His2, Pall ForteBio, Fremont, CA). Studies were performed at 25 °C with shaking at 1000 rpm in PBS supplemented with 2 mM Mg²⁺ and 10 μM Ca²⁺. Sensor tips were hydrated in buffer for 30 min prior to use. The 96-microwell plates were filled with 200 μL of buffer or samples and incubated for 10 min prior measurements for system stabilization. Camk2α (3.5 μg/mL) was loaded on the His2 sensor for 120 s and washed for 60 s. A reference sensor without Camk2α served as a background control. Association and dissociation phases (300 s each) were monitored at various concentrations of Fbp2 protein ranging from 50 to 400 nM. Kinetic parameters were determined by global fitting with the 1:1 model. Response values from the last 10 s of the association phase were averaged and used for equilibrium dissociation constants calculation. Data were analyzed with ForteBio Data Analysis 11.0 software (Pall ForteBio, San Jose, CA, USA).