title: Preparation of filamentous actin for polarized total internal reflection fluorescence microscopy (polTIRFM) motility assays.
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. In this protocol, filamentous actin (F-actin) is polymerized from purified, monomeric actin (G-actin) for use in polTIRFM motility assays in which actin interacts with myosin. The procedures include (1) the preparation of unlabeled F-actin from G-actin; (2) the preparation of F-actin that is sparsely labeled with 6'-IATR (6'-iodoacetamidotetramethylrhodamine); and (3) the preparation of F-actin with a combination of unlabeled, biotinylated, and rhodamine-labeled monomers. Rhodamine-phalloidin actin, also used in polTIRFM assays, can be prepared using a procedure similar to the one for unlabeled actin.
authors.forename: John F
authors.initials: JF
authors.lastname: Beausang
authors.forename: Yujie
authors.initials: Y
authors.lastname: Sun
authors.forename: Margot E
authors.initials: ME
authors.lastname: Quinlan
authors.forename: Joseph N
authors.initials: JN
authors.lastname: Forkey
authors.forename: Yale E
authors.initials: YE
authors.lastname: Goldman
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D005456:Fluorescent Dyes / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D009068:Movement; D009218:Myosins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D013048:Specimen Handling / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D000199:Actins; D005456:Fluorescent Dyes; D009218:Myosins