Tetracysteine FLASH diacetate 2 mM DMSO Solution

FLASH diacetate is a cell-permeable version of FlASH, which 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 Diacetate working solution
Incubate the cells with FLASH Diacetate 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.

FLASH Diacetate 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 Diacetate 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 Diacetate 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

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Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
Tetracysteine FLASH Reagent 2 mM DMSO Solution	498	517	80000¹	0.7900¹, 0.95²	0.32	0.35

References

View all 24 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

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

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

Using Tetracysteine-Tagged TDP-43 with a Biarsenical Dye To Monitor Real-Time Trafficking in a Cell Model of Amyotrophic Lateral Sclerosis.
Authors: Ng, Janice S W and Hanspal, Maya A and Matharu, Naunehal S and Barros, Teresa P and Esbjörner, Elin K and Wilson, Mark R and Yerbury, Justin J and Dobson, Christopher M and Kumita, Janet R
Journal: Biochemistry (2019): 4086-4095

The novel mitochondria localization of influenza A virus NS1 visualized by FlAsH labeling.
Authors: Tsai, Chuan-Fu and Lin, Hsin-Yi and Hsu, Wei-Li and Tsai, Ching-Hsiu
Journal: FEBS open bio (2017): 1960-1971

Page updated on December 17, 2024

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Physical properties

Molecular weight	748.56
Solvent	DMSO

Spectral properties

Absorbance (nm)	487
Correction Factor (260 nm)	0.32
Correction Factor (280 nm)	0.35
Extinction coefficient (cm ^-1 M ^-1)	80000¹
Excitation (nm)	498
Emission (nm)	517
Quantum yield	0.7900¹, 0.95²

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501