title: Site-specific fluorescent labeling of tubulin.
abstract: Fluorescent tubulin can be prepared in which a fluorophore is covalently bound to the protein at only the carboxy terminus of the α-subunit of the αβ-tubulin dimer. This two-step procedure consists of an enzymatic reaction followed by a bioorthogonal chemical reaction. In the first step of the process, the enzyme tubulin tyrosine ligase is used to attach a reactive tyrosine derivative, 3-formyltyrosine, to the protein. In the second step of the procedure, a fluorophore possessing a complementary reactive functional group, such as a hydrazine, hydrazide, or hydroxylamine, is allowed to react with the protein under conditions that are compatible with native tubulin. Polymerization-competent, fluorescently labeled tubulin can be prepared in just a few hours using this protocol. The method described here should be useful for attaching virtually any probe or material to tubulin at this site.
authors: Department of Chemistry, Binghamton University, State University of New York, Binghamton, New York, USA.
authors: Kamalika
authors: K
authors: Mukherjee
authors: Susan L
authors: SL
authors: Bane
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology; D002417:Cattle; D005456:Fluorescent Dyes; D010453:Peptide Synthases / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D014404:Tubulin / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D014404:Tubulin; D010453:Peptide Synthases; C023213:tyrosyltubulin ligase
keywords: Bioorthogonal chemistry; Fluorescence; Site-specific protein labeling; Tubulin; Tubulin tyrosine ligase; Tyrosine derivatives; Unnatural amino acid