Protocol

Biarsenical Labeling of Tetracysteine-Tagged Proteins for Tracking Existing and Newly Synthesized Pools of Proteins

Department of Veterinary and Biomedical Sciences and Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583-0900, USA

¹Corresponding author (apattnaik2{at}unl.edu).

INTRODUCTION

Genetically tagging proteins of interest with fluorescent labels such as green fluorescent protein is a valuable technique for studying protein localization and interactions in live cells. However, the relatively large size of fluorescent fusion proteins can limit the utility of this approach, and sometimes the tag can render the protein of interest nonfunctional. Small genetic tagging with tetracysteine (TC) and subsequent labeling with membrane-permeable biarsenical dyes can overcome these limitations. This protocol describes the method to detect TC-tagged proteins by biarsenical labeling in live cells. A small amino acid tag containing four cysteines (CCPGCC) is introduced to a protein of interest. Cells expressing proteins with this tag are then treated with membrane-permeable dyes (FlAsH-EDT₂ or ReAsH-EDT₂), and the two arsenic groups of the dyes each bind to the two thiols in the TC motif. Once bound, FlAsH and ReAsH are converted to a fluorescent state (green and red fluorescence at excitation maxima of 508 and 593 nm, respectively) and can be detected by fluorescence microscopy. The method is particularly suitable for investigating protein dynamics that are difficult to study using a fluorescent protein tag. It also is a powerful method for visualizing trafficking of newly synthesized proteins in real time, because existing and newly synthesized pools of proteins can be labeled differentially with the red and green dyes. A method for labeling virally produced proteins is also presented.