4.3 SPR studies of L-hIGFBP2 and its mutants
SPR studies to estimate the binding affinity of IGF-1 and IGF-2 to L-h IGFBP2 and its mutant (lacking the residues 150-161) were carried out on a Biacore 3000 instrument. IGF-1 (10 μg/μL) in 10 mM sodium acetate buffer, pH 4.0, was immobilized at a flow rate of 2 μL/min onto the activated CM-5 sensor chip using the amine coupling kit. 520 response units (RU) of IGF-1 were coupled on separate flow cells. The immobilization of pure IGF-2 (5.5 μg/μL) was carried out on a separate chip at 25 °C at a flow rate of 2 μL/min and 350 response units of IGF-2 were coupled on the flow cell. Binding experiments were carried out for different concentrations of L-h IGFBP2 and its mutants in HBS buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20, pH 7.5) at a flow rate of 10 uL/min. Regeneration of the biosensor surface before the next reading was done by passing 5 μL of 2 M MgCl2 across the chip. The data were fit to the Langmuir 1:1 binding model using the fitting procedures in BIA-evaluation software 3·0·1 to calculate dissociation constants.