Fig. 3.
Fig. 3. Kinetic parameters for binding of factor VIII light chain to immobilized scFv. / Factor VIII light chain (20 nM) was incubated with immobilized scFv KM33 (3.3 fmol/mm2), KM35 (63.3 fmol/mm2), KM36 (67.5 fmol/mm2), KM37 (28.3 fmol/mm2), KM38 (32.3 fmol/mm2), and KM41 (25.0 fmol/mm2) in 200 mM NaCl, 2 mM CaCl2, 0.05% (v/v) Tween 20, and 20 mM HEPES, pH 7.4 at a flow rate of 20 μL/min for 2 minutes at 25°C. Dissociation was initiated on replacement of ligand solution by buffer. Response is indicated as resonance units (RU) and is corrected for nonspecific binding (< 5%). To obtain similar values of resonance units for all scFv, the amount of scFv KM33 immobilized was approximately 10-fold reduced compared to the other scFv. Similar values of kon and koffwere found when higher concentrations of scFv KM33 were immobilized.

Kinetic parameters for binding of factor VIII light chain to immobilized scFv.

Factor VIII light chain (20 nM) was incubated with immobilized scFv KM33 (3.3 fmol/mm2), KM35 (63.3 fmol/mm2), KM36 (67.5 fmol/mm2), KM37 (28.3 fmol/mm2), KM38 (32.3 fmol/mm2), and KM41 (25.0 fmol/mm2) in 200 mM NaCl, 2 mM CaCl2, 0.05% (v/v) Tween 20, and 20 mM HEPES, pH 7.4 at a flow rate of 20 μL/min for 2 minutes at 25°C. Dissociation was initiated on replacement of ligand solution by buffer. Response is indicated as resonance units (RU) and is corrected for nonspecific binding (< 5%). To obtain similar values of resonance units for all scFv, the amount of scFv KM33 immobilized was approximately 10-fold reduced compared to the other scFv. Similar values of kon and koffwere found when higher concentrations of scFv KM33 were immobilized.

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