5-IAF (5-Iodoacetamidofluorescein)

A fluorescent probe for labeling proteins

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货号: ajce52602
CAS: 63368-54-7
别名: 5-碘乙酰氨基荧光素; 5-Iodoacetamidofluorescein
分子式: C22H14INO6
分子量: 515.25
纯度: >98%
溶解度: DMF: 30 mg/ml,DMSO: 30 mg/ml,DMSO:PBS (pH 7.2) (1:3): 0.25 mg/ml,Ethanol: 500μ g/ml
储存: Store at -20°C
库存: 现货

Background

5-IAF is a fluorescent probe for labeling proteins.¹ It has been used to label proteins through a reaction with sulfhydryl groups. 5-IAF displays excitation/emission maxima of 491/518 nm, respectively, which can shift during protein labeling or changes in hydrophobicity and can be quenched by potassium iodide. 5-IAF has been used to monitor ligand binding and conformational changes of the Na⁺/K⁺-ATPase.²

1.Hartig, P.R., Bertrand, N.J., and Sauer, K.5-Iodoacetamidofluorescein-labeled chloroplast coupling factor 1: Conformational dynamics and labeling-site characterizationBiochemistry16(19)4275-4282(1977) 2.Kapakos, J.G., and Steinberg, M.Ligand binding to (Na,K)-ATPase labeled with 5-iodoacetamidofluoresceinJ. Biol. Chem.261(5)2084-2089(1986)

Protocol

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].

参考文献:

[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.