Protocol for Preparation and purification of Cyanine3 NHS ester -Protein(pro-FD):Dried dyes(Cyanine3 NHS ester) were dissolved to 1 mg/ml in anhydrous DMF and stored frozen in aliquots.
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.
Cyanine3 NHS ester (non-sulfonated) 可用作反应性染料,用于标记生物分子中的氨基。该试剂通常用于标记可溶性蛋白\多肽\寡核苷酸或 DNA。Cy3 NHS ester ?(non-sulfonated)需要使用有机共溶剂(例如 DMF 或 DMSO和乙腈)用于标记[1,2]。
Cy3 (Cyanine 3) 是一种发橘黄色荧光的花青素荧光染料 它的荧光很亮，荧光位于橙色区域(~550nm 激发, ~570 nm 发射),对pH不敏感,并且处在肉眼较敏感的可见光区,在绝大多数荧光仪器上都可使用。Cy3 可以通过配有标准的TRITC滤波器的荧光计\成像仪及显微镜检测。由于其固有的高消光系数,甚至肉眼也可以很容易地检测到凝胶和溶液中的 Cy3[3,4]。
NHS活化酯(N-羟基琥珀酰亚胺酯,N-hydroxysuccinimide ester, succinimidyl ester)是生物标记反应中最常用的活化基团 NHS活化Cy3分子中的羧基,让它可以和目标生物分子上的胺基(主要伯胺)反应生成稳定的酰胺键,从而把Cy3分子标记到生物大分子上 由于游离胺基是蛋白,抗体,多肽表面的常见官能团(来自于赖胺酸侧链),Cy3-NHS可以和它们直接反应 核酸分子中天然不带游离胺基,只有胺基修饰过的RNA或者DNA才可以和Cy3-NHS反应[5,6]。
Cy3-NHS活化酯固体可在-20℃保存一年,溶解后(无论水溶液还是无水有机溶剂)应尽快使用,不能长期保存。
参考文献:
[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.
Protocol for Preparation and purification of??Cyanine3 NHS ester –Protein（pro-FD）：
Dried dyes(Cyanine3 NHS ester) were dissolved to 1 mg/ml in anhydrous DMF and stored frozen in aliquots.
1.Labeling reactions were performed in 20 mM NaH2PO4?and 300 mM NaCl (pH 6.8) buffer with 10:1 dye(Cyanine3 NHS ester) to protein molar ratio and incubated in the dark for 4 h at room temperature and then overnight at 4℃. The reactions were stopped by adding 1/10 volume 1 M Tris-HCl (pH 8) buffer, and after 30-fold dilution with ice-cold equilibration buffer, samples were applied to a 5 55-mm YMC-BioPro S30 column equilibrated with 20 mM NaH2PO4?(pH 6.8) buffer and eluted with a 0 350 mM NaCl linear gradient in the same buffer. Single-labeled Cy3 pro-FD had an A549/A280?ratio of 4.
2.Fractions were assayed homogeneity by SDS-PAGE, and then selected fractions containing single-labeled proteins were combined and concentrated on 10 kDa cutoff concentrators to 1 mg/ml and stored frozen in aliquots.
3.The concentrations of Cy3 pro-FD was calculated based on the extinction coefficients of Cy3 (ε549nm = 150,000 M?-1cm?-1) and molecular masses of 26.5 kDa.
This protocol only provides a guideline, and should be modified according to your specific needs.
参考文献:
[1].Gábor Oroszlán, Elod Kortvely, et,al. MASP-1 and MASP-2 Do Not Activate Pro–Factor D in Resting Human Blood, whereas MASP-3 Is a Potential Activator: Kinetic Analysis Involving Specific MASP-1 and MASP-2 Inhibitors. J Immunol 15 January 2016; 196 (2): 857–865.
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