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. 2012 May 1;2012(5):10.1101/pdb.prot069369 pdb.prot069369.
doi: 10.1101/pdb.prot069369.

Fluorescent labeling of myosin V for polarized total internal reflection fluorescence microscopy (polTIRFM) motility assays

John F Beausang, Yujie Sun, Margot E Quinlan, Joseph N Forkey, Yale E Goldman

Fluorescent labeling of myosin V for polarized total internal reflection fluorescence microscopy (polTIRFM) motility assays

John F Beausang et al. Cold Spring Harb Protoc. .

Abstract

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes how to exchange bifunctional rhodamine-calmodulin (BR-CaM) for wild-type calmodulin (WT-CaM) on the lever arm of myosin V. BR-CaM is exchanged at low stoichiometry (∼0.4 BR-CaM per double-headed myosin V) to obtain myosin V molecules with one BR-CaM and to limit the proportion of myosin V molecules with two or more probes. The stoichiometry is very sensitive to the concentration of calcium during the exchange reaction. The labeled myosin V can subsequently be used for investigating the motility of myosin V in vitro with a polTIRFM processive motility assay, which is performed on substrate-attached actin.

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References

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