Fluorescent labeling of tetracysteine-tagged proteins in intact cells

Abstract

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.

A Perspective on Studying G-Protein–Coupled Receptor Signaling with Resonance Energy Transfer Biosensors in Living Organisms

“…Popular pairs are two genetically encoded fluorescent proteins in case of FRET (predominantly cyan and yellow variants derived from Aequorea victoria green fluorescent protein [GFP]) or one luminescent protein (frequently luciferase from Renilla reniformis) and one fluorescent protein (frequently variant forms of the GFP from Aequorea victoria) in case of BRET. An alternative combination has been realized by employing a small tetracysteine tag, to which a fluorescein derivative (i.e., FlAsH, fluorescein arsenical hairpin binder) can bind with high specificity as an acceptor (Hoffmann et al, 2010). Other tags (such as SNAP-, CLIP-and Halo-tags) are also available (Comps-Agrar et al, 2011).…”

A Tetracysteine-tag and HeLa Cell System for the Dynamic Analysis of the Localization and Gating Properties of a Specific Connexin Isoform

“…The expression of Cx43-TC in HeLa cells was detected by staining with FlAsH (Toronto Research Chemicals Inc.) according to the procedure developed by Hoffmann et al 25 and Crivat et al 26 with slight modification. Briefly, a 500 nM FlAsH solution was added to the confluent culture of HeLa cells for incubation under a 5% CO 2 condition for 1 h at 37°C.…”

Trafficking of Bluetongue Virus Visualized by Recovery of Tetracysteine-Tagged Virion Particles

“…The tagged proteins are specifically recognized by membrane-permeant biarsenical dyes that fluoresce when bound to the cysteine pairs in the TC motif. In addition, the differential labeling of the tagged proteins with two fluorescent biarsenical dyes, FlAsH (green) and ReAsH (red) (18,19), makes this technology a powerful tool for the real-time visualization of nascent protein synthesis and trafficking in cells. To date, this technology has been used successfully in enveloped viruses (21)(22)(23)(24) but not for any complex capsid virus such as BTV.…”

“…Since the BTV genome consists of 10 segmented dsRNA molecules, each approximately 0.8 to 3.9 kb, the capacity of each segment to accommodate for-eign genes is limited (17). Hence, as an alternate strategy, we used the biarsenical-tetracysteine (TC) technology, which involves the use of small TC tags with a CCPGCC motif, which can be inserted into a protein without the risk of disrupting the overall structure of the targeted protein (18)(19)(20). The tagged proteins are specifically recognized by membrane-permeant biarsenical dyes that fluoresce when bound to the cysteine pairs in the TC motif.…”