Techniques Used in the Moore Lab
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|We use a variety of techniques to study the biochemical and mechanical properties of molecular motors, the cytoskeleton and biopolymers.
Total internal reflectance fluorescence microscopy
The unloaded motility assay gives important information about the motion generating capacity of molecular motors. However, motor molecules also produce force. We assess the effects of myosin or thin filament regulatory protein mutations on relative (to wild-type) force generation. In this assay, load is applied to the myosin motor by applying it to the surface along with small amounts of non motile actin binding proteins (e.g., alpha-actinin). The alpha-actinin will introduce a frictional load that resists filament motion. The amount of alpha-actinin required to stop filament motion is an indicator of myosin force generating capability. Since the load is applied to all of the filaments in a field of view, this assay is useful for rapidly screening a variety of experimental conditions (e. g.: regulated actin, unregulated actin, submaximal calcium activation, etc.).
Optical traps use photon momentum to capture and hold small transparent particles with a higher refractive index than the medium. As a trapped particle moves away form the laser focus there is a restoring force that is proportional to the distance the particle moves. Therefore, an optical trap can be used as a very sensitive force tranducer that can measure forces in the piconewton range.
Laser trap loading assay
We use both optical trap and frictional loading based assays to measure the inherent force generating capacity of mutant myosin molecules. Frictional loading assays provide an indication of relative force while optical trap based assays measure the absolute isometric force generating capability of the myosin mutants one filament at a time.