Kinase experiment:

HepG2 cells are lysed in 1 mL 2% acetonitrile in water, and transfered to a 1.5 mL centrifuge tube. An additional 200 μL 2% acetonitrile is used to rinse the dish and combined with the cell extract. To measure the free GSH concentration, three aliquots of 80 μL cell extract are immediately transferred to 0.5 mL centrifuge tubes after a quick mix. Then 20 μL 50 μM NAC is added, followed by the addition of 100 μL 400 μM 5-IAF to the mixture. The derivatization is conducted at room temperature in dark for 1 h, after which the mixtures are centrifuged at 9000× g for 5 min to sediment the insoluble proteins. 10 μL supernatant is diluted 100× with water and analyzed immediately by CE-LIF[2].

5-IAF is an idoacetamide derivate of fluoresceine.

8 µM GSH sample solution is derivatized with 8, 80, 200, 400, 800, 1600, 2000, 2500 µM 5-IAF, respectively and analyzed with CE-LIF. The highest signal intensity is obtained with 400 and 800 µM 5-IAF[2].

[1]. Salvatore Sotgia, et al. Plasma L-Ergothioneine Measurement by High-Performance Liquid Chromatography and Capillary Electrophoresis after a Pre-Column Derivatization with 5-Iodoacetamidofluorescein (5-IAF) and Fluorescence Detection. PLoS One. 2013; 8(7): e70374. [2]. Yan Wang, et al. Determination of free and protein-bound glutathione in HepG2 cells using capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr A. 2009 Apr 17;1216(16):3533-7.