Figure 2. CHO-K1 cells expressing a tetracysteine-tagged version of β-tubulin labeled with TC-ReAsH reagent. Upon treatment with vinblastine, a compound known to perturb cytoskeletal structure, tubulin drastically rearranges from a reticular structure (left) to rod shaped (right).
FlAsH and ReAsH—Tetracysteine-Based Protein Detection
The smallest available expression tag and protein-labeling technology based on the affinity and specificity of the green FlAsH and red ReAsH reagents to a six-amino acid motif.
How it works
The biarsenical labeling reagents FlAsH-EDT2 and ReAsH-EDT2 become fluorescent when they bind to recombinant proteins containing the tetracysteine (TC) motif Cys-Cys-Pro-Gly-Cys-Cys (Figure 1). FlAsH and ReAsH technology provides sensitive live cell imaging and subcellular localization of proteins containing the TC-tag by using fluorescence microscopy.
How it has been used
- Protein localization, turnover, and trafficking
- Receptor signaling and internalization
- Correlation of light microscopy with electron microscopy
- Pulse-chase and double-labeling experiments
- Enzyme activity studies
- SDS-PAGE detection of tagged proteins
- Affinity purification
What it offers
The TC-FlAsH/ReAsH expression tag-based fluorescence labeling technology was developed for live-cell imaging. It is ideal for protein localization or real-time protein production studies, though its versatility offers a range of benefits:
- Rapid protein detection—easily constructed six-amino acid tags
- Multiplexing flexibility—choice of red or green fluorescence from the same tagged protein
- Small tag size—reduces interference with target protein
- Stable but non-covalent binding— allows dual labeling, pulse-chase experiments and other dynamic, real-time studies
- Compatible with live or fixed cell imaging and multiplexing
- Multiple applications beyond direct labeling for imaging
- Affinity purification
Roger Tsien and colleagues first described the use of biarsenical reagents for site-specific protein labeling in live cells in 1998. The biarsenical labeling technology works through the high-affinity interaction of arsenic for thiols. FlAsH is a fluorescein derivative, modified to contain two arsenic atoms at a set distance from each other. ReAsH is based on resorufin and has been similarly modified. FlAsH and ReAsH are virtually non-fluorescent when bound to ethane dithiol (EDT). When FlAsH-EDT or ReAsH-EDT bind to tetracysteine (TC) sequences, EDT is displaced and the tags become highly fluorescent in green or red, respectively. The most commonly used tetracysteine is the six amino acid Cys-Cys-Pro-Gly-Cys-Cys sequence. As this sequence rarely appears in endogenous proteins, incorporating the sequence into target proteins generates a small but highly specific target for protein labeling.
Since Tsien and colleagues’ publication, many applications for biarsenical reagents have been described. Most FlAsH and ReAsH applications focus on labeling specific proteins in live cells (Figure 2). Tagged proteins are rapidly labeled without fixation or laborious antibody labeling protocols and the small tag is unlikely to interfere with protein function. Protein localization is thus conveniently tracked. In addition, using more than one label makes it feasible to study dynamic processes such as protein turnover or assembly in pulse-chase experiments. Other applications of the Invitrogen Molecular Probes products such as TC-FlAsH and TC-ReAsH technology include affinity purification, SDS-PAGE protein detection, and analysis of protease activity. The BAL wash buffer replaces the previously supplied wash buffers and yield superior signal to noise.
|Original description of FlAsH as a tool for site-specific labeling of proteins in living cells||Griffin BA, Adams SR, Tsien RY (1998) Science 281:269|
|Affinity purification of tetracysteine tagged proteins||Thorn KS, Naber N, Matuska M et al. (2000) Protein Science 9:213|
|Correlation of electron microscopy to fluorescence microscopy||Gaietta G, Deerinck TJ, Adams SR et al. (2002) Science 296:503|
|Improved photostable FRET-competent TC probes||Spagnuolo CC, Vermeij RJ, Jares-Erijman EA (2006) JACS 128:12040|
|Live cell imaging of synthetic peptides expressed in plants||Estévez JM, Somerville C (2006) BioTechniques 41:569|