Cy3 NHS ester (non-sulfonated)

Cyanine3 NHS ester (non-sulfonated) can be used as a reactive dye to label the amino group in a biological molecule. This reagent is commonly used to label soluble protein polypeptide oligonucleotide or DNA.Cy3 NHS ester (non-sulfonated) requires the use of an organic co-solvent (such as DMF or DMSO and acetonitrile) for the labeling^[1,2].

Cy3 (Cyanine 3) is a kind of orange fluorescence anthocyanin fluorescence dye its fluorescence is very bright, fluorescence is located in the orange region (~550nm excitation, ~570 nm emission), not sensitive to pH, and in the naked eye more sensitive visible light region, in the vast majority of fluorescence instruments can be used.Cy3 can be detected by fluorimeter imager and microscope equipped with standard TRITC filters.Cy3 in gels and solutions can be easily detected even by the naked eye due to its inherently high extinction coefficient^[3,4]

NHS activated ester (N-hydroxysuccinimide, n-hydroxysuccinimide ester, succinimidyl ester (NHS), the most commonly used activating group in biomarker reactions, activates the carboxyl group in Cy3 molecules, allowing it to react with the amine group (main primary amine) on the target biomolecules to form a stable amide bond, thus labeling Cy3 molecules to biomolecules. Since free amine groups are common functional groups on the surface of proteins, antibodies and polypeptides (from the side chain of lysamine acid),Cy3-NHS can react directly with them. Nucleic acid molecules naturally do not contain free amine groups, and only RNA or DNA modified with amine groups can react with Cy3-NHS^[5,6].

Cy3-NHS activated ester solid can be stored at -20℃ for one year, after dissolution (whether aqueous or anhydrous organic solvent) should be used as soon as possible, not long term storage.

References:

[1]. Gao XQ, Han JX,et,al. Identification of the differential expressive tumor associated genes in rectal cancers by cDNA microarray. World J Gastroenterol. 2007 Jan 21;13(3):341-8. doi: 10.3748/wjg.v13.i3.341. PMID: 17230600; PMCID:?PMC4065886.

[2]. Reid, D. A, Keegan, S, et,al.Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair. Proceedings of the National Academy of Sciences of the U.S.A., 2015, 112(20).

[3]. Cho Y, et al. Mechanism of Cyanine5 to Cyanine3 Photoconversion and Its Application for High-Density Single-Particle Tracking in a Living Cell. J Am Chem Soc. 2021 Sep 8;143(35):14125-14135.

[4]. Li H, et al. Silver enhanced ratiometric nanosensor based on two adjustable Fluorescence Resonance Energy Transfer modes for quantitative protein sensing. Biosens Bioelectron. 2017 Jan 15;87:428-432.?

[5]. Groszewicz, et al. N-Hydroxysuccinimide-activated esters as a functionalization agent for amino cellulose: synthesis and solid-state NMR characterization. Cellulose 27, 1239–1254 (2020). https://doi.org/10.1007/s10570-019-02864-5

[6]. Li M. Optimization of N-hydroxysuccinimide ester coupling with aminoallyl-modified RNA for fluorescent labeling. Bioengineered. 2020 Dec;11(1):599-606. doi: 10.1080/21655979.2020.1765487. PMID: 32449472; PMCID: PMC8291868.