Surface Plasmon Resonance (SPR)

Real-time binding assays were performed on a Biacore 3000 Surface Plasmon Resonance (SPR) instrument (GE Healthcare, Milan, Italy). Full-length recombinant proteins, Hsp90, Hsp70, HOP, and Hsp40 (what about p23??) were immobilized at similar immobilization levels (1503, 2410, 2172, 3100 RU, respectively) on a CM5 Biacore sensor chip, at ∼100μg/mL in 10 mM sodium acetate, pH 5.0, by using the EDC/NHS chemistry, with a flow rate of 2 μL/min and an injection time of 7 min. Binding assays were carried out by injecting 90 μL of the analyte, at 30 μL/min, with HBS (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, pH 7.4), and 1% (v/v) DMSO as running-buffer. The BIAevaluation analysis package (version 4.1, GE Healthcare), was used to subtract the signal of the reference channel, with a DMSO correction procedure.