title: An assay to measure the affinity of proteins for microtubules by quantitative fluorescent microscopy.
abstract: We report a fluorescence-based assay for measuring the affinity of microtubule binding proteins for microtubules. The affinity of any fluorescently tagged protein for taxol-stabilized microtubules can be measured with this assay. We describe the assay and provide a detailed protocol. Using this assay, we found that the affinity of the Dam1 complex for microtubules is decreased by the presence of free unpolymerized tubulin and is sensitive to the salt concentration in the binding buffer. These effects may account for the previous differences in binding affinities reported.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA 98115, USA.
authors.forename: Beth
authors.initials: B
authors.lastname: Graczyk
authors.forename: Trisha N
authors.initials: TN
authors.lastname: Davis
mesh_terms: D008856:Microscopy, Fluorescence / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D008870:Microtubules / Q000737:chemistry*; D017239:Paclitaxel / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D014404:Tubulin / Q000737:chemistry; D014894:Weights and Measures
chemical_list: D008869:Microtubule-Associated Proteins; D011506:Proteins; D014404:Tubulin; D017239:Paclitaxel