Carboxyl-polystyrene beads of 2.0-μm diameters (Spherotech, Lake Forest, IL) were covalently coupled with streptavidin (Invitrogen) as previously described.[15 (link), 17 (link)] Briefly, streptavidin-coated beads were incubated with a VWF domain fragment. SpyCatcher was coupled to biotin-DNA handle, and then incubated with streptavidin beads. For pulling experiments, one bead with an Avi-His-SpyTag VWF fragment on its surface was fixed by the micropipette, while the other bead coupled with SpyCatcher-DNA handle was trapped and controlled by the optical tweezers. The force measurement was performed at force-ramp mode, where the stretching force increases linearly with the pulling distance. The force-extension data were fitted to the WLC model [18 (link)] to determine its mechanical properties.
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Measurement of VWF Fragment Mechanics
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Corresponding Organization : University of Kansas Medical Center
Other organizations : Lehigh University
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- Carboxyl-polystyrene beads of 2.0-μm diameters (Spherotech, Lake Forest, IL) covalently coupled with streptavidin (Invitrogen)
- VWF domain fragment incubated with streptavidin-coated beads
- SpyCatcher coupled to biotin-DNA handle and incubated with streptavidin beads
- Stretching force increases linearly with the pulling distance during force-ramp mode
- Mechanical properties of the VWF fragment determined by fitting the force-extension data to the WLC model
- Streptavidin-coated beads
- SpyCatcher-DNA handle
- Avi-His-SpyTag VWF fragment
- Micropipette and optical tweezers for bead manipulation and force measurement
- No positive controls explicitly mentioned
- No negative controls explicitly mentioned
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