Alexa fluor 647 NHS ester TEA

Alexa Fluor 647 NHS ester TEA is a widely used fluorescent dye containing an activated carboxyl group for labeling substrates with primary amines (R-NH₂), such as proteins, oligonucleotides, and nucleic acids, and it has a maximum excitation wavelength of 650nm and a maximum emission wavelength of 665nm^[1-2]. N-hydroxysuccinimide (NHS) esters can undergo nucleophilic substitution reactions with proteins that contain an N-terminal cysteine, forming new amide bonds and thus enabling the labeling of proteins^[3]. Alexa Fluor 647 NHS ester TEA performs well under Direct Stochastic Optical Reconstruction Microscopy (dSTORM) imaging conditions and is widely used for cell bioimaging and super-resolution microscopy^[4]. Under microscope imaging conditions, the reversible photo-induced isomerization of Alexa Fluor 647 NHS ester TEA forms two dark species, which is related to the blinking in dSTORM. Alexa Fluor 647 NHS ester TEA used in super-resolution fluorescence microscopy undergo light-induced “blinking”, enabling the localization of fluorophores with spatial resolution beyond the optical diffraction limit. The permanent photobleaching quantum yield of Alexa Fluor 647 NHS ester TEA is very low and can be partially suppressed by an anaerobic redox buffer^[5]. Alexa Fluor 647 NHS ester TEA can also be used in combination with labeling reagents such as phalloidin for the staining of cell lines, as well as for the staining of giant unilamellar vesicles (GUVs)^[6-7].

References:
[1] Anderson GP, Nerurkar NL. Improved fluoroimmunoassays using the dye Alexa Fluor 647 with the RAPTOR, a fiber optic biosensor. J Immunol Methods. 2002;271(1-2):17-24.
[2] Wuethrich I, Guillen E, Ploegh HL. A mouse monoclonal antibody against Alexa Fluor 647. Monoclon Antib Immunodiagn Immunother. 2014;33(2):109-120.
[3] Jiang H, D'Agostino GD, Cole PA, Dempsey DR. Selective protein N-terminal labeling with N-hydroxysuccinimide esters. Methods Enzymol. 2020;639:333-353.
[4] Fan S, Webb JEA, Yang Y, et al. Observing the Reversible Single Molecule Electrochemistry of Alexa Fluor 647 Dyes by Total Internal Reflection Fluorescence Microscopy. Angew Chem Int Ed Engl. 2019;58(41):14495-14498.
[5] Karlsson JKG, Laude A, Hall MJ, Harriman A. Photo-isomerization of the Cyanine Dye Alexa-Fluor 647 (AF-647) in the Context of dSTORM Super-Resolution Microscopy. Chemistry. 2019;25(65):14983-14998.
[6] Moghal MMR, Islam MZ, Sharmin S, Levadnyy V, Moniruzzaman M, Yamazaki M. Continuous detection of entry of cell-penetrating peptide transportan 10 into single vesicles. Chem Phys Lipids. 2018;212:120-129.
[7] Bharadwaj A, Kumar A, Mitra R, Jaganathan BG, Boruah BR. Enhanced fluorescence blinking of AF647 fluorophores in Mowiol via violet and UV light induced recovery for superior localization microscopy. Methods Appl Fluoresc. 2024;12(3):10.1088/2050-6120/ad4ae6.

Alexa Fluor 647 NHS ester TEA是一种广泛使用的荧光染料，有可用于标记含有伯胺（R-NH₂）的底物（如蛋白质、寡核苷酸和核酸）的活化羧基，其最大激发波长为650nm，最大发射波长为665nm^[1-2]。N-羟基琥珀酰亚胺（NHS）酯可以与含有N端半胱氨酸的蛋白质发生亲核取代反应，形成新的酰胺键，从而实现对蛋白质的标记^[3]。Alexa Fluor 647 NHS ester TEA在直接随机光学重建显微镜（dSTORM）成像条件下表现出色，广泛用于细胞生物成像和超分辨显微镜^[4]。在显微镜成像条件下，Alexa Fluor 647 NHS ester TEA的可逆光诱导异构化会形成两种暗态物种，这与dSTORM中的闪烁有关。在超分辨荧光显微镜中使用的Alexa Fluor 647 NHS ester TEA会发生光诱导的“闪烁”，从而能够实现超越光学衍射极限的空间分辨率对荧光团进行定位。Alexa Fluor 647 NHS ester TEA 的永久光漂白量子产率非常低，并且可以通过厌氧氧化还原缓冲液部分抑制^[5]。Alexa Fluor 647 NHS ester TEA 还可以用于与鬼笔环肽等标记试剂结合，用于细胞系的染色，以及对大单层囊泡（GUVs）进行染色^[6-7]。