Regulated in vitro Motility Assay

Regulated Motility

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________pCa 10 (no Calcium) _____________pCa 4 (saturating Calcium)______

In addition to the standard motility assay, the regulated in vitro motility assay includes the two regulatory proteins Troponin and Tropomyosin and the cation Calcium.  These proteins are incubated in solution with the fluorescent actin filaments and self assemble about the filaments so that their orientation is just as they are in vivo.  Troponin can bind Calcium causing a conformational change that moves Tropomyosin and opens a binding site for Myosin. Therefore regulated motility involves acto-myosin interactions that are dependent upon the free Calcium to open binding sites and allow force production.  Different calcium concentrations as well as physiological conditions are injected into the flow cell and the resultant motility can display velocity dependent upon Calcium concentration as well as myosin’s Calcium sensitivity.  This more complicated version of the motility assay is a closer match to the real physiological function occurring in vivo