FlAsH-EDT2

Labeling proteins tagged with tetracysteine using FlAsH-EDT2^[1]
This protocol only provides a guideline, and should be modified according to your specific needs.
Cell Labeling:
1. Wash the cells twice with 2ml of HBSS/glucose solution.
2. Transfer 1ml of the HBSS/glucose solution for each sample to a Falcon tube.
3. Take 50-100µl of the HBSS/glucose solution and add the HBSS/glucose solution to the FlAsH-EDT2 solution, mixing well.
4. Add the mixed solution to the Falcon tube, ensuring the solution is homogeneous.
5. Add 1ml of this solution to each culture dish, gently swirling the dish to ensure initial mixing.
6. Incubate at 37°C (with a humidified atmosphere and CO₂ as appropriate) for 1h. During the labeling process, the concentrations in the system are 500nM FlAsH-EDT2, 12.5µM EDT (1,2-ethanedithiol), and 0.1% (v/v) DMSO.
Removal of Excess and Nonspecifically Bound Dye:
7. Preparation of the washing solution: Prepare a 500mM EDT solution in DMSO.
8. Wash the cells with HBSS/glucose solution containing 250µM EDT for 10min.
9. Wash the cells 2-3 times with HBSS/glucose solution (without EDT) to reduce EDT residue.
10. Repeat the washing step 1 or 2 times to further reduce background fluorescence.
Imaging:
11. Confocal microscopy: Directly excite FlAsH using a 514nm argon laser and record its fluorescence emission.

References:
[1] Hoffmann C, Gaietta G, Zürn A, et al. Fluorescent labeling of tetracysteine-tagged proteins in intact cells. Nat Protoc. 2010 Sep;5(10):1666-77.