Figure 5
Figure 5. The binding of inactivated factor Xa (Xai) and factor Va to immobilized myosin were determined using BLI and the Octet Red system. Binding studies using BLI were conducted with factor Xai, DG-factor Xai, factor Va, and factor Xai in the presence of factor Va and factor IXai. Proteins were in buffer containing 50 mM Tris (pH 7.4), 0.6 mM MgCl2, 5 mM CaCl2, 0.005% Tween 20, and 0.1% PEG, 300 mM NaCl at room temperature. Skeletal muscle myosin was captured onto the Octet Red probe surface by monoclonal anti–myosin heavy chain (MF20) antibodies, and then after washing, the various proteins were loaded. The protein association and dissociation time courses were then monitored by Octet Red for 30 minutes at room temperature. There was negligible nonspecific binding of inactivated factor Xai or DG-factor Xai or factor Va to anti–myosin antibody coated sensors in the absence of myosin. Octet RED analysis software (ForteBio) was used to analyze the data from sensorgram data. The y-axis indicates the change in optical interference which is a result of the binding response. (A) Sensorgrams depicting the binding of factor Xai to immobilized myosin (ligand concentrations from top to bottom: 120, 60, 30, 15, and 7.5 nM). (B) Sensorgrams depicting the binding of DG-factor Xai to immobilized myosin (ligand concentrations from top to bottom: 240, 120, 60, 30, 15, and 7.5 nM). (C) Sensorgrams depicting the binding of factor Va to immobilized myosin (ligand concentrations from top to bottom: 30, 15, 7.5, and 3.8 nM). (D) Sensorgrams depicting the binding of factor Xai to myosin (ligand concentrations from top to bottom: 120, 60, 30, 15, and 7.5 nM) in the presence of 10 nM factor Va. (E) Sensorgrams depicting the absence of binding of 120 nM factor IXai to immobilized myosin. The solid black lines indicate best fits of the experimental data for association and dissociation reactions and were used to obtain values for kon and koff and calculation of Kd. In the presence of 5 mM EDTA and absence of Ca++ and Mg++ ions, neither factor Xai nor DG-factor Xai detectably bound to myosin (data not shown).

The binding of inactivated factor Xa (Xai) and factor Va to immobilized myosin were determined using BLI and the Octet Red system. Binding studies using BLI were conducted with factor Xai, DG-factor Xai, factor Va, and factor Xai in the presence of factor Va and factor IXai. Proteins were in buffer containing 50 mM Tris (pH 7.4), 0.6 mM MgCl2, 5 mM CaCl2, 0.005% Tween 20, and 0.1% PEG, 300 mM NaCl at room temperature. Skeletal muscle myosin was captured onto the Octet Red probe surface by monoclonal anti–myosin heavy chain (MF20) antibodies, and then after washing, the various proteins were loaded. The protein association and dissociation time courses were then monitored by Octet Red for 30 minutes at room temperature. There was negligible nonspecific binding of inactivated factor Xai or DG-factor Xai or factor Va to anti–myosin antibody coated sensors in the absence of myosin. Octet RED analysis software (ForteBio) was used to analyze the data from sensorgram data. The y-axis indicates the change in optical interference which is a result of the binding response. (A) Sensorgrams depicting the binding of factor Xai to immobilized myosin (ligand concentrations from top to bottom: 120, 60, 30, 15, and 7.5 nM). (B) Sensorgrams depicting the binding of DG-factor Xai to immobilized myosin (ligand concentrations from top to bottom: 240, 120, 60, 30, 15, and 7.5 nM). (C) Sensorgrams depicting the binding of factor Va to immobilized myosin (ligand concentrations from top to bottom: 30, 15, 7.5, and 3.8 nM). (D) Sensorgrams depicting the binding of factor Xai to myosin (ligand concentrations from top to bottom: 120, 60, 30, 15, and 7.5 nM) in the presence of 10 nM factor Va. (E) Sensorgrams depicting the absence of binding of 120 nM factor IXai to immobilized myosin. The solid black lines indicate best fits of the experimental data for association and dissociation reactions and were used to obtain values for kon and koff and calculation of Kd. In the presence of 5 mM EDTA and absence of Ca++ and Mg++ ions, neither factor Xai nor DG-factor Xai detectably bound to myosin (data not shown).

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