Cy5 NHS Ester | AAT Bioquest

Cyanine 5 monosuccinimidyl ester

equivalent to Cy5® NHS ester

Cy5 NHS Ester is a far-red fluorescent labeling reagent that efficiently conjugates to amine groups on proteins and peptides, providing excellent tissue penetration and minimal background for in vivo imaging applications.

Far-red fluorescence: Deep tissue penetration with minimal autofluorescence for in vivo imaging applications
633 nm laser compatible: Optimized for standard red laser lines in confocal microscopy and flow cytometry
High-efficiency protein labeling: NHS ester chemistry provides 80–95% conjugation yields with antibodies and proteins
Alternative to Alexa Fluor 647: Spectrally equivalent with improved cost-effectiveness for large-scale labeling projects

Cy5 spectrum. Cy5 (Cyanine-5) is a fluorescent compound with an excitation peak at 651 nm and an emission peak at 670 nm. Other spectra of interest include: FITC (Fluorescein-5-isothiocyanate), Cy3, Cy7, Alexa Fluor 647 and iFluor 647. Cy5 (Cyanine-5) belongs to the following categories: Amine Reactive Dyes and Probes for Conjugation, Förster Resonance Energy Transfer (FRET) and Real-Time PCR (qPCR).

Protocol

Catalog	Size	Price	Quantity
151	1 mg	Price
151-5MG	5 mg	Price

Overview

Cy5® NHS Ester (GE Healthcare) and Cyanine5 NHS ester are chemically equivalent, far-red fluorescent dyes used for labeling peptides, proteins and oligonucleotides, with an excitation wavelength of 651 nm and an emission wavelength of 670 nm.

Background

Cy5 is a far-red fluorescent dye belonging to the cyanine dyes family, which contains other popular fluorophores such as Cy3 (yellow) and Cy7 (near infrared). Cy5 is sometimes also sold under the names, Cyanine5 or Sulfo-Cy5; these are all the same chemical structure.

The “sulfo” in the name derives from the SO3- groups present in the molecule, which improves its water solubility. Non-sulfonated Cy5, which lacks these groups, is less soluble and more likely to form aggregates in solution. Because of this, the sulfonated form of Cy3 is far more common than the non-sulfonated form.

Usage

Cy5 NHS ester has a molecular weight of 855.07 daltons. It is provided as a blue powder, with triethylammonium salts instead of potassium salts, for improved reconstitution in DMSO and DMF.

The NHS ester functional group enables easy conjugation to primary amines, such as the lysine residues of antibodies and amine-modified oligonucleotides. Cy5 is also available in maleimide form, for conjugation to the sulfide groups of proteins (e.g. cysteine), as well as phosphoramidite form, for usage in oligonucleotide synthesis.

Excitation and emission

Cy5 has an excitation peak at 651 nm and an emission peak at 670 nm. It is well excited by the 633 nm or 647 nm laser lines. For detection, a fluorescence channel such as the Cy5 filter set or a 660/20 bandpass filter can be used.

Applications

Cy5 can be conjugated to antibodies and used in flow cytometry. For example, Cy5-CD antibody conjugates are useful for the screening of cell surface markers and sorting by fluorescence (FACS). Cy5 can also be labeled to oligonucleotides, for use in real-time PCR techniques, such as in TaqMan probes.

Alternatives

Cy7 is a near-infrared alternative to Cy5 that has an excitation and emission of 756 nm and 779 nm, respectively.

iFluor® 647 is a spectrally similar fluorophore that improves upon water solubility and brightness, while at a lower cost.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Cyanine 5 monosuccinimidyl ester [equivalent to Cy5® 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	116.949 µL	584.747 µL	1.169 mL	5.847 mL	11.695 mL
5 mM	23.39 µL	116.949 µL	233.899 µL	1.169 mL	2.339 mL
10 mM	11.695 µL	58.475 µL	116.949 µL	584.747 µL	1.169 mL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
mg	÷	855.07 g/mol	=	mL	x	mM	=	mmol

Citations

View all 33 citations: Citation Explorer

Molecular profiling of the single-cell proteome via gel electrophoresis and 3D single-molecule imaging

Authors:

Kamarulzaman, Latiefa and Kim, Sooyeon and Hidaka, Takuya and Tsuchida, Misaki and Taniguchi, Yuichi

Journal:

Nature Communications (2026): 5727

Comprehensive molecular profiling of single-cell proteome via gel electrophoresis and 3D single-molecule imaging

Authors:

Kamarulzaman, Latiefa and Kim, Sooyeon and Hidaka, Takuya and Tsuchida, Misaki and Taniguchi, Yuichi

Journal:

bioRxiv (2026): 2026--01

Munc18 modulates syntaxin phase separation to promote exocytosis

Authors:

Pei, Qing and Chen, Qixin and Tian, Zhiqi and Zhu, Le and Chen, Yang and Gong, Jihong and Wang, Shen and Xiang, Yijuan and Khamo, John S and Fan, Jiaqi and others,

Journal:

Nature Neuroscience (2025): 1--9

BAR-PH tandem of ACAP4 remodels membranes to drive migration via Ezrin-dependent activation

Authors:

Huang, Shan and Chen, Jiasheng and Wang, Xinyu and Song, Xiaoyu and Zhu, Xiaojie and Fu, Chuanhai and Zhang, Xuan and Liu, Xing and Wang, Chao

Journal:

Journal of Molecular Cell Biology (2025): mjaf038

mInsc coordinates Par3 and NuMA condensates for assembly of the spindle orientation machinery in asymmetric cell division

Authors:

Huang, Shijing and Fu, Minjie and Gu, Aihong and Zhao, Ruiqian and Liu, Ziheng and Hua, Wei and Mao, Ying and Wen, Wenyu

Journal:

International Journal of Biological Macromolecules (2024): 135126

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	855.07
Solvent	DMSO

Spectral properties

Correction factor (260 nm)	0.02
Correction factor (280 nm)	0.03
Correction factor (482 nm)	0.009
Correction factor (565 nm)	0.09
Extinction coefficient (cm ^-1 M ^-1)	250000 ¹
Excitation (nm)	651
Emission (nm)	670
Quantum yield	0.27 ¹ , 0.4 ²

Usage and storage

Intended use	Research Use Only (RUO)
Storage	Freeze (< -15 °C); Minimize light exposure
CAS	146368-14-1

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Page updated on September 27, 2024