title: Labeling tetracysteine-tagged proteins with biarsenical dyes for live cell imaging.
abstract: Correlation of real-time or time-lapse light microscopy (LM) with electron microscopy (EM) of cells can be performed with biarsenical dyes. These dyes fluorescently label tetracysteine-tagged proteins so that they can be imaged with LM and, upon fluorescent photoconversion of 3,3'-diaminobenzidine tetrahydrochloride (DAB), with EM as well. In the following protocol, cells expressing tetracysteine-tagged proteins are labeled for 1 h with biarsenical dyes. The volumes indicated are for a single 30-mm culture dish containing 2 mL of labeling medium. Scale the suggested volumes up or down depending upon the size of the culture dish used in the labeling. The same procedure can be adapted for longer labeling times by lowering the amount of dye used to 50-100 nM; however, the amount of the competing dithiol EDT is maintained at 10-20 μM. Longer labeling times often produce higher signal-to-noise ratios and cause less trauma to the treated cells prior to imaging.
authors.forename: Guido M
authors.initials: GM
authors.lastname: Gaietta
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Deerinck
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
mesh_terms: D001152:Arsenicals; D002460:Cell Line; D004396:Coloring Agents; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008853:Microscopy / Q000379:methods; D011994:Recombinant Proteins / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D001152:Arsenicals; D004396:Coloring Agents; D011994:Recombinant Proteins