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. 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
FIAsH EDT Flourescent

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

Technology

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 (see above image). 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.
FIAsH ReAsH Detection

Applications

Since Tsien and colleagues’ publication, many applications for biarsenical reagents have been described (link to applications). Most FIAsH and ReAsH applications focus on labeling specific proteins in live cells. 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 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.

Table 1 - Reference List

Year Applications and Articles Reference
1998 Original description of FlAsH as a tool for site-specific labeling of proteins in living cells Griffin (1998) Science 281:269
2000 Affinity purification of tetracysteine tagged proteins Thorn (2000) Protein Science 9:213
2002 Correlation of electron microscopy to fluorescence microscopy Gaietta (2002) Science 296:503
2006 Improved photostable FRET-competent TC probes Spagnuolo (2006) JACS 128:12040
2006 Live cell imaging of synthetic peptides expressed in plants Estevez (2006) BioTechniques 41:569