Development of fluorescent tags for labeling newly synthesized proteins

BIOL 15

John D Fisk, fisk@cheme.caltech.edu1, Kimberly E. Beatty, beatty@caltech.edu1, Jeremy M. Baskin, baskin@berkeley.edu2, Carolyn R Bertozzi, bertozzi@cchem.berkeley.edu2, and David A. Tirrell, tirrell@caltech.edu1. (1) Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd, MC 210-41, Pasadena, CA 91125, (2) Department of Chemistry, University of California, Berkeley, B84 Hildebrand Hall, #1460, Bertozzi Group, Berkeley, CA 94720-1460
The fluorescent labeling of proteins is an indispensable tool in biology. The use of non-canonical amino acids as specific bioorthogonal handles to identify, select, and label newly synthesized proteins has recently been demonstrated. Here we describe the development of tags consisting of fluorescent dyes conjugated to cyclooctyne bearing moieties. These dye conjugates specifically react with azide containing non-canonical amino acids without toxic copper catalysis. Various coumarin, fluorescein and BODIPY dye conjugates are discussed. The conjugates were investigated for their ability to label proteins expressed in the presence and absence of non-canonical amino acid in fixed mammalian cells. Both confocal microscopy and FACS were used to identify and quantify dye labeling efficiency. Additional experiments involving labeling in live cells are also described.
 

Frontiers in Chemical Biology
5:00 PM-7:00 PM, Sunday, August 19, 2007 BCEC -- Exhibit Hall - B2, Poster

Division of Biological Chemistry

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007