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Tetracysteine FLASH Reagent *2 mM DMSO Solution*
FLASH is a fluorescein derivative, modified to contain two arsenic atoms at a set distance from each other. It was developed by Roger Tsien and colleagues in 1998. The biarsenical labeling technology works through the high-affinity interaction of arsenic for thiols. When FlASH binds to tetracysteine (TC) sequences, its biarsenical group reacts rapidly with Cys-Cys moiety and the tag becomes highly fluorescent in green. The biarsenical labeling reagent FLASH is the smallest expression tag for labeling a protein that contains a six-amino acid motif with a Cys-Cys-X1-X2-Cys-Cys amino acid sequence. 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. FLASH generates a strong green fluorescent signal when binding to recombinant proteins containing the tetracysteine motif Cys-Cys-Pro-Gly-Cys-Cys. It can be used for monitoring protein localization, turnover and trafficking, receptor signaling and internalization.
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells
- Prepare FLASH Reagent working solution
- Incubate the cells with FLASH Reagent working solution for 15-60 minutes
- Image the cells using the FITC filter set
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
Note Make single used aliquots and store at - 20 °C. Avoid freeze and thaw cycle.
FLASH Reagent stock solution (800X)
Add 67 µL of DMSO to the vial and mix well.Note Make single used aliquots and store at - 20 °C. Avoid freeze and thaw cycle.
PREPARATION OF WORKING SOLUTION
FLASH Reagent working solution (1X)
Dilute the 800X stock solution at 1:800 in an appropriate buffer such as serum-free or low-level serum (~1%) medium, HHBS, or Opti-MEM® medium to make a 1X labeling solution and mix well.Note Make the working solution just before use.
Note For cells transduced with lentivirus, a 0.5X working solution may be optimal. Depending on the levels of specific and background fluorescent signal, you can optimize the working solution to better visualize your labeled protein. We recommend trying a concentration range of 0.4 to 4X working solution.
Table 1.The following table is the suggested volume of labeling solution to use for different tissue culture formats.
| Plate | 96-well | 48-well | 24-well | 12-well | 6-well |
| Volume | 100 µL | 150 µL | 250 µL | 500 µL | 1 mL |
SAMPLE EXPERIMENTAL PROTOCOL
- Prepare cells as desired.
- Remove the growth medium from the cells and wash cells once with an appropriate medium.
- Add the appropriate amount of 1X FLASH Reagent working solution to each well (See table for the appropriate volume).
Note Appropriately discard any unused 1X working solution according to your institution’s guidelines. Do not reuse the 1X working solution. - Incubate the cells at room temperature for 15-60 minutes, protected from light.
- Wash the cells with 250 µM BAL in serum free medium or buffer of your choice 2 times.
- Image your labeled protein using a fluorescence microscope with a FITC filter set.
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
| Tetracysteine REASH Reagent *2 mM DMSO Solution* | 571 | 584 | 650001 | 0.751 | - | - |
| Tetracysteine FLASH diacetate *2 mM DMSO Solution* | 498 | 517 | 800001 | 0.79001, 0.952 | 0.32 | 0.35 |
References
View all 50 references: Citation Explorer
Fluorescent labelling of membrane fatty acid transporter CD36 (SR-B2) in the extracellular loop.
Authors: Liu, Yilin and Rodriguez-Calvo, Ricardo and Wang, Shujin and Zhu, Xiaoqing and Broers, Jos L V and Glatz, Jan F C and Luiken, Joost J F P and Neumann, Dietbert
Journal: PloS one (2019): e0210704
Authors: Liu, Yilin and Rodriguez-Calvo, Ricardo and Wang, Shujin and Zhu, Xiaoqing and Broers, Jos L V and Glatz, Jan F C and Luiken, Joost J F P and Neumann, Dietbert
Journal: PloS one (2019): e0210704
Probing Arrestin Function Using Intramolecular FlAsH-BRET Biosensors.
Authors: Strungs, Erik G and Luttrell, Louis M and Lee, Mi-Hye
Journal: Methods in molecular biology (Clifton, N.J.) (2019): 309-322
Authors: Strungs, Erik G and Luttrell, Louis M and Lee, Mi-Hye
Journal: Methods in molecular biology (Clifton, N.J.) (2019): 309-322
Probing the Effect of Sildenafil on Progesterone and Testosterone Production by an Intracellular FRET/BRET Combined Approach.
Authors: Casarini, Livio and Riccetti, Laura and Limoncella, Silvia and Lazzaretti, Clara and Barbagallo, Federica and Pacifico, Salvatore and Guerrini, Remo and Tagliavini, Simonetta and Trenti, Tommaso and Simoni, Manuela and Sola, Marco and Di Rocco, Giulia
Journal: Biochemistry (2019): 799-808
Authors: Casarini, Livio and Riccetti, Laura and Limoncella, Silvia and Lazzaretti, Clara and Barbagallo, Federica and Pacifico, Salvatore and Guerrini, Remo and Tagliavini, Simonetta and Trenti, Tommaso and Simoni, Manuela and Sola, Marco and Di Rocco, Giulia
Journal: Biochemistry (2019): 799-808
Small Fluorescein Arsenical Hairpin-Based Förster Resonance Energy Transfer Analysis Reveals Changes in Amino- to Carboxyl-Terminal Interactions upon OAG Activation of Classical Transient Receptor Potential 6.
Authors: Fiedler, Susanne and Storch, Ursula and Erdogmus, Serap and Gudermann, Thomas and Mederos Y Schnitzler, Michael and Dietrich, Alexander
Journal: Molecular pharmacology (2019): 90-98
Authors: Fiedler, Susanne and Storch, Ursula and Erdogmus, Serap and Gudermann, Thomas and Mederos Y Schnitzler, Michael and Dietrich, Alexander
Journal: Molecular pharmacology (2019): 90-98
Fluorometric detection of protein-ligand engagement: The case of phosphodiesterase5.
Authors: Di Rocco, Giulia and Martinelli, Ilaria and Pacifico, Salvatore and Guerrini, Remo and Cichero, Elena and Fossa, Paola and Franchini, Silvia and Cardarelli, Silvia and Giorgi, Mauro and Sola, Marco and Ponterini, Glauco
Journal: Journal of pharmaceutical and biomedical analysis (2018): 335-342
Authors: Di Rocco, Giulia and Martinelli, Ilaria and Pacifico, Salvatore and Guerrini, Remo and Cichero, Elena and Fossa, Paola and Franchini, Silvia and Cardarelli, Silvia and Giorgi, Mauro and Sola, Marco and Ponterini, Glauco
Journal: Journal of pharmaceutical and biomedical analysis (2018): 335-342
Page updated on December 17, 2024
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