Cyanine3 NHS Ester | AAT Bioquest

Cyanine 3 monosuccinimidyl ester

equivalent to Cy3® NHS ester

Cyanine 3 monosuccinimidyl ester is an amine-reactive orange fluorescent dye that forms stable amide bonds with proteins and antibodies, widely used for creating bright, photostable bioconjugates for microscopy and flow cytometry.

Orange fluorescence: Provides bright, photostable signal optimal for 532 nm laser excitation in microscopy and flow cytometry
Amine-reactive conjugation: Forms stable amide bonds with lysine residues at pH 8.5 for efficient protein and antibody labeling
Multicolor imaging applications: Compatible with FITC and Cy5 for triple-labeling experiments in immunofluorescence and FISH

Cy3 spectrum. Cy3 (Cyanine-3) is a fluorescent compound with an excitation peak at 555 nm and an emission peak at 569 nm. Other spectra of interest include: mFluor™ Violet 420, Alexa Fluor 555, and XFD555 *Alexa Fluor® 555 Equivalent*. Cy3 (Cyanine-3) belongs to the following categories: Antibody and Protein Labeling, and Immunophenotyping.

Catalog	Size	Price	Quantity
141	1 mg	Price

Overview

Cy3® NHS Ester (GE Healthcare) and Cyanine 3 monosuccinimidyl ester are chemically equivalent, reactive dyes used for fluorescence labeling biological molecules by targeting amino-groups.

Background

Cy3 is a yellow fluorescent dye belonging to the cyanine dyes family. It is sometimes also referred to as Sulfo-Cy3; the two are chemically equivalent. In contrast, non-sulfonated Cy3 does not have SO3- groups. Consequently, non-sulfonated Cy3 has lower water solubility than Cy3, as well as increased likelihood of forming aggregates in squeous solution. For this reason, sulfo-Cy3 and Cy3 are much more common than non-sulfonated Cy3.

Usage

Cy3 NHS ester has a molecular weight of 829.03 daltons. It is supplied as a red powder and can be reconstituted using DMSO. The NHS ester functional group allows for easy conjugation to targets with primary amine groups, such as the lysine residue of proteins and antibodies or amine-modified oligonucleotides. Labeling reactions create a covalent bond between the dye and biomolecule; for the best efficiency, the reaction should occur in a buffer with pH = 8.5 ± 0.5. Once reacted, a degree of labeling (DOL) should be calculated for the conjugate. This is important as overlabeling (i.e. high DOL) can result in self-quenching of the Cy3 fluorohpore.

Excitation / Emission

Cy3 has two important wavelengths: an excitation peak at 555 nm and an emission peak at 569 nm. When excited by a suitable light source, such as a 532 nm or 555 nm laser, it will emit yellow fluorescence. As such, compatible fluorescence channels include Cy3 or TRITC filter sets. Cy3 fluorescence is fairly resistant to changes in pH, tolerating pH 4 to pH 10.

Applications

Cy3 can be conjugated to antibodies and used for immunofluorescence (IF) and immunocytochemistry (ICC) applications. For example, cells treated with mouse anti-tubulin primary antibodies, followed by Cy3 goat anti-mouse IgG secondary antibodies, can be used to visualize cytoskeletal structures.

A second application is the use of Cy3 to label oligonucleotides. In fluorescence in situ hybridization (FISH), Cy3-oligo probes allow for spatial mapping of genetic material in cells. Combined with a quencher, Cy3-oligo conjugates can be used as TaqMan probes (or molecular beacons) for real-time PCR techniques.

Other formats

Cy3 is also available with maleimide moieties. This allows for conjugation to proteins and antibodies which present sulfhydryl-groups, such as cysteine residues. For incorporation of Cy3 into an oligonucleotide during chemical synthesis, Cy3 phosphoramidites can be used. In this process, phosphoramidites are added from the 3' to 5' direction.

Cy5 is an alternative to Cy3 that belongs to the same family for fluorescent dyes. It has an excitation wavelength of 651 nm and an emission wavelength of 670 nm.

Cy3B is an analog of Cy3 that is more resistant to internal de-excitation, thus resulting in a measured increase in fluorescence signal and improved photostability.

iFluor® 555 is a spectrally similar fluorophore that offers improved water solubility and brightness, with decreased photobleaching and cost.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Cyanine 3 monosuccinimidyl ester [equivalent to Cy3® NHS ester] to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	120.623 µL	603.114 µL	1.206 mL	6.031 mL	12.062 mL
5 mM	24.125 µL	120.623 µL	241.246 µL	1.206 mL	2.412 mL
10 mM	12.062 µL	60.311 µL	120.623 µL	603.114 µL	1.206 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
mg	÷	829.03 g/mol	=	mL	x	mM	=	mmol

Citations

View all 33 citations: Citation Explorer

CLASP-mediated competitive binding in protein condensates directs microtubule growth

Authors:

Jia, Xuanyan and Lin, Leishu and Guo, Siqi and Zhou, Lulu and Jin, Gaowei and Dong, Jiayuan and Xiao, Jinman and Xie, Xingqiao and Li, Yiming and He, Sicong and others,

Journal:

Nature Communications (2024): 6509

Competitive binding-mediated mesoscale protein-protein interactions direct microtubule growth

Authors:

Wei, Zhiyi and Jia, Xuanyan and Lin, Leishu and Guo, Siqi and Zhou, Lulu and Jin, Gaowei and Dong, Jiayuan and Xiao, Jinman and Xie, Xingqiao and Li, Yiming and others,

The disordered protein SERF promotes $\alpha$-Synuclein aggregation through liquid-liquid phase separation

Authors:

Liu, He-Ning and Wang, Ting and Hu, Jin-Jian and Chen, Long and Shi, Xiangyan and Li, Yan-Mei and Luo, Shi-Zhong

Journal:

Journal of Biological Chemistry (2024): 105667

Manganese regulation of COPII condensation controls circulating lipid homeostasis

Authors:

Wang, Xiao and Huang, Runze and Wang, Yawei and Zhou, Wenjing and Hu, Yating and Yao, Yuanhang and Cheng, Kunlun and Li, Xin and Xu, Bolin and Zhang, Jie and others,

Journal:

Nature Cell Biology (2023): 1--14

Temporal and spatial assembly of inner ear hair cell ankle link condensate through phase separation

Authors:

Wang, Huang and Du, Haibo and Ren, Rui and Du, Tingting and Lin, Lin and Feng, Zhe and Zhao, Dange and Wei, Xiaoxi and Zhai, Xiaoyan and Wang, Hongyang and others,

Journal:

Nature Communications (2023): 1657

References

View all 21 references: Citation Explorer

Excitation of Cy5 in self-assembled lipid bilayers using optical microresonators

Authors:

Freeman LM, Li S, Dayani Y, Choi HS, Malmstadt N, Armani AM.

Journal:

Appl Phys Lett (2011): 143703

Theranostic cRGD-BioShuttle Constructs Containing Temozolomide- and Cy7 For NIR-Imaging and Therapy

Authors:

Wiessler M, Hennrich U, Pipkorn R, Waldeck W, Cao L, Peter J, Ehemann V, Semmler W, Lammers T, Braun K.

Journal:

Theranostics (2011): 381

Rational approach to select small peptide molecular probes labeled with fluorescent cyanine dyes for in vivo optical imaging

Authors:

Berezin MY, Guo K, Akers W, Livingston J, Solomon M, Lee H, Liang K, Agee A, Achilefu S.

Journal:

Biochemistry (2011): 2691

In vivo detection of embryonic stem cell-derived cardiovascular progenitor cells using Cy3-labeled Gadofluorine M in murine myocardium

Authors:

Adler ED, Bystrup A, Briley-Saebo KC, Mani V, Young W, Giovanonne S, Altman P, Kattman SJ, Frank JA, Weinmann HJ, Keller GM, Fayad ZA.

Journal:

JACC Cardiovasc Imaging (2009): 1114

Thiazole orange and Cy3: improvement of fluorescent DNA probes with use of short range electron transfer

Authors:

Menacher F, Rubner M, Berndl S, Wagenknecht HA.

Journal:

J Org Chem (2008): 4263

Physical properties

Molecular weight	829.03
Solvent	DMSO

Spectral properties

Correction factor (260 nm)	0.07
Correction factor (280 nm)	0.073
Extinction coefficient (cm ^-1 M ^-1)	150000 ¹
Excitation (nm)	555
Emission (nm)	569
Quantum yield	0.15 ¹

Storage, safety and handling

Certificate of Origin	Download PDF
H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501
CAS	146368-16-3

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Page updated on September 5, 2024