Surface plasmon resonance analysis of fibulin-1C and C1 esterase inhibitor with calumenin and reticulocalbin.

<p>(A) Reticulocalbin was immobilised to the chip and various concentrations of GST-fibulin-1C was applied at indicated concentrations in buffer (10 mM Hepes, 150 mM NaCl, 5 mM CaCl₂, 1 mM EGTA and 0.005% Tween 20, pH 7.4). The dissociation constant is estimated to be approx. 60 nM. GST was also applied to the chip and showed no detectable binding. (B) Various concentrations of GST-fibulin-1C were applied at indicated concentrations in buffer with EDTA (10 mM Hepes, 150 mM NaCl, 20 mM EDTA and 0.005% Tween 20 pH 7.4). The dissociation constant is estimated to be approx. 50 nM. (C) GST-fibulin-1C, 0.8 μM, was applied to a chip with immobilised GST-calumenin and to a chip with immobilised GST. The affinity of GST-fibulin-1C was estimated to approx. 50 nM. The buffer was similar to that given in A. (D) C1 esterase inhibitor was applied in varying concentrations to a chip with immobilized reticulocalbin. The dissociation constant was estimated to be approx. 1 μM. The buffer was similar to that given in A. (E) GST-calumenin was immobilized to a chip and C1 esterase inhibitor was applied in varying concentrations as indicated. The dissociation constant was estimated to approx. 150 nM. The buffer was similar to that given in A. (F) C1 esterase inhibitor was applied to the chip in buffer containing 4 mM free Ca²⁺ (10 mM Hepes, 150 mM NaCl, 5 mM CaCl₂, 1 mM EGTA and 0.005% Tween 20, pH 7.4). In the dissociation phase the Ca²⁺ concentration was varied from 3.5 mM to 0.1 mM as indicated. No detectable binding was observed at 0.1 mM Ca²⁺. Regeneration of the sensor chips after each analysis cycle was performed with 10 mM glycine, 20 mM EDTA, 500 mM NaCl and 0.005% Tween 20, pH 4.0.