Fluorescent labeling of tetracysteine-tagged proteins in intact cells.
In this paper, we provide a general protocol for labeling proteins with the membrane-permeant fluorogenic biarsenical dye fluorescein arsenical hairpin binder-ethanedithiol (FlAsH-EDT₂). Generation of the tetracysteine-tagged protein construct by itself is not described, as this is a protein-specific process. This method allows site-selective labeling of proteins in living cells and has been applied to a wide variety of proteins and biological problems. We provide here a generally applicable labeling procedure and discuss the problems that can occur as well as general considerations that must be taken into account when designing and implementing the procedure. The method can even be applied to proteins with expression below 1 pmol mg⁻¹ of protein, such as G protein-coupled receptors, and it can be used to study the intracellular localization of proteins as well as functional interactions in fluorescence resonance energy transfer experiments. The labeling procedure using FlAsH-EDT₂ as described takes 2-3 h, depending on the number of samples to be processed.
Hoffmann
Carsten
C
Department of Pharmacology and Toxicology, University of Würzburg, Germany.
Gaietta
Guido
G
Zürn
Alexander
A
Adams
Stephen R
SR
Terrillon
Sonia
S
Ellisman
Mark H
MH
Tsien
Roger Y
RY
Lohse
Martin J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001152:Arsenicals / Q000737:chemistry; D001665:Binding Sites; D002478:Cells, Cultured; D005452:Fluoresceins / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D009942:Organometallic Compounds / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*
C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; D001152:Arsenicals; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D011506:Proteins