Measuring p53-FGF1 Interactions via SPR

Interactions between recombinant proteins were measured by surface plasmon resonance (SPR) using a Biacore 3000 instrument. (GE Healthcare) at 25 ℃. The recombinant human full-length p53 and its DNA-binding domain (p53_DBD) dissolved in 10 mM sodium acetate, pH 5.0 were immobilized on CM5 sensor chip surface (GE Healthcare) at about 1000 RU and 800 RU, respectively, using an amine coupling protocol. To determine interaction parameters between FGF1 and p53 or p53_DBD, measurements were performed in 10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, 0.1% BSA, 0.02% NaN₃, pH 7.4. Recombinant FGF1 protein at the concentrations ranging from 16 to 2048 nM was injected at a flow of 30 μl/min. The association and disassociation phases were monitored for 4 min and 5 min, respectively. After each measurement the sensor was regenerated with 2.5 M NaCl and 10 mM NaOH solution. The data were analyzed using the BIAevaluation 4.1 software (GE Healthcare). Equilibrium dissociation constant (K_D) was calculated from fitted saturation binding curve [25 (link)]. Response values from the last 10 s of the association phase were averaged and used to determine the K_D.