Cm5 sensor chip surface

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.

The interaction measurements were performed using a Biacore 3000 instrument (GE Healthcare) at 25°C in PBS with 0.05% Tween20, 0.1% BSA, 0.02% NaN₃, and pH 7.4. The extracellular domains of FGFR1 in Fc fusions (in 10 mM sodium acetate, pH 5.0) were immobilized on the CM5 sensor chip surface (GE Healthcare) at 8800 RU using an amine coupling protocol. To determine kinetic constants of the interaction between peptibodies F4_1 or F4_2 or F4_3 and FGFR1, a set of dilutions of peptibodies at concentrations ranging from 0.15 to 4.8 μM or FGF4 (concentration from 75 to 600 nM) were injected at a flow of 30 μl/min. The association and disassociation were monitored for 180 and 240 s, respectively. Between injections, 10 mM glycine (pH 1.5) was applied to regenerate the sensor chip surface. The data were analyzed using the BIAevaluation 4.1 software (GE Healthcare). SPR measurements showed that the peptibodies did not interact 1:1 with the receptor, and therefore, the data did not fit to the standard 1:1 Langmuir binding model. For this reason, we used steady-state affinity analysis (which is particularly suitable for measurements of weaker interactions) to determine the K_D value without computing kon and koff (Suh et al., 2014 (link)). Response values from the last 10 s of the association phase were averaged and used to determine the K_D.