Kinesin Motility Assays
We use a number of microscopic assays to characterize the movement of kinesin motors along microtubules. In the microtubule gliding assay, motors are adsorbed to a glass substrate and the fluorescently labeled microtubules are visualized moving over the bed of motors.
Inverting the geometry, we can immobilize microtubules on the surface and attach the motors to micron-scale glass beads. These beads can be visualized moving along the microtubules. Additionally, these beads can be trapped with an optical tweezer, which is a focused laser that is introduced into the microscope objective. The momentum of the light traps the beads at the focus of the beam and when provides a restoring force when motors try to pull the bead out of the trap.
The third assay we use is the single-molecule fluorescence assay. Motors are labeled with green fluorescent protein or another fluorophore and microtubules are labeled with a different fluorophore and attached to the surface of a glass coverslip. The sample is illuminated by total internal reflection fluorescence (TIRF) imaging, which limits the illumination to the ~100 nm closest to the surface. Using a sensitive enhanced CCD camera, single motor molecules can be visualized moving along the microtubules.