Cyanine 7 monosuccinimidyl ester [equivalent to Cy7® NHS ester]

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	881.11
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.05
Correction Factor (280 nm)	0.036
Correction Factor (482 nm)	0.0005
Correction Factor (565 nm)	0.0193
Correction Factor (650 nm)	0.165
Extinction coefficient (cm ^-1 M ^-1)	250000
Excitation (nm)	756
Emission (nm)	779
Quantum yield	0.3

Storage, safety and handling

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

Overview SDS Protocol

CAS

477908-53-5

Molecular weight

881.11

Correction Factor (260 nm)

0.05

Correction Factor (280 nm)

0.036

Correction Factor (482 nm)

0.0005

Correction Factor (565 nm)

0.0193

Correction Factor (650 nm)

0.165

Extinction coefficient (cm ^-1 M ^-1)

250000

Excitation (nm)

756

Emission (nm)

779

Quantum yield

0.3

A variety of cyanine 7 (Cy7®) dyes has been used to label biological molecules for fluorescence imaging and other fluorescence-based biochemical analysis. They are widely used for labeling peptides, proteins and oligos etc. Cy7® dye conjugates are one type of the most common near infrared red fluorophores used in in vivo imaging applications. Cy7® NHS ester readily reacts with amino groups. AAT Bioquest offers Cy dye NHS esters in the form of triethylammonium salts that are more soluble in DMSO and DMF than the corresponding potassium salts that are offered by some other vendors. The Cy dye triethylammonium salts have the same reactivity and give the conjugates identical to the the Cy dye potassium salts. Cy7® is the trademark of GE Healthcare.

Example protocol

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Protein stock solution (Solution A)

Mix 100 µL of a reaction buffer (e.g., 1 M sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g. antibody, protein concentration >2 mg/mL if possible) to give 1 mL protein labeling stock solution. Note: The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer. Note: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation. Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results. Note: The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.

2. Cyanine 7 monosuccinimidyl ester stock solution (Solution B)

Add anhydrous DMSO into the vial of Cyanine 7 monosuccinimidyl ester to make a 10 mM stock solution. Mix well by pipetting or vortex. Note: Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with Cyanine 7 monosuccinimidyl ester. You might need further optimization for your particular proteins. Note: Each protein requires distinct dye/protein ratio, which also depends on the properties of dyes. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity.

Run conjugation reaction

Use 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point: Add 5 µL of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) into the vial of the protein solution (95 µL of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD. Note: We recommend to use 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too less or too high, determine the optimal dye/protein ratio at 5:1, 15:1 and 20:1 respectively.
Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.

Purify the conjugation

The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.

Prepare Sephadex G-25 column according to the manufacture instruction.
Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate. Note: For immediate use, the dye-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses. Note: For longer term storage, dye-protein conjugate solution need be concentrated or freeze dried.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Cyanine 7 monosuccinimidyl ester [equivalent to Cy7® 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	113.493 µL	567.466 µL	1.135 mL	5.675 mL	11.349 mL
5 mM	22.699 µL	113.493 µL	226.986 µL	1.135 mL	2.27 mL
10 mM	11.349 µL	56.747 µL	113.493 µL	567.466 µL	1.135 mL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

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Spectral properties

Correction Factor (260 nm)	0.05
Correction Factor (280 nm)	0.036
Correction Factor (482 nm)	0.0005
Correction Factor (565 nm)	0.0193
Correction Factor (650 nm)	0.165
Extinction coefficient (cm ^-1 M ^-1)	250000
Excitation (nm)	756
Emission (nm)	779
Quantum yield	0.3

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)	Correction Factor (482 nm)	Correction Factor (565 nm)
Cyanine 3 monosuccinimidyl ester [equivalent to Cy3® NHS ester]	555	569	150000¹	0.15¹	0.07	0.073	-	-
Cyanine 5 monosuccinimidyl ester [equivalent to Cy5® NHS ester]	651	670	250000¹	0.27¹, 0.4²	0.02	0.03	0.009	0.09
Cyanine 7 bissuccinimidyl ester [equivalent to Cy7® bisNHS ester]	756	779	250000	0.3	0.05	0.036	0.0005	0.0193

Images

Figure 1. Chemical structure for Cyanine 7 monosuccinimidyl ester [equivalent to Cy7® NHS ester]

Citations

View all 18 citations: Citation Explorer

Highly sensitive magnetic particle imaging of vulnerable atherosclerotic plaque with active myeloperoxidase-targeted nanoparticles
Authors: Tong, Wei and Hui, Hui and Shang, Wenting and Zhang, Yingqian and Tian, Feng and Ma, Qiang and Yang, Xin and Tian, Jie and Chen, Yundai
Journal: Theranostics (2021): 506

Highly sensitive magnetic particle imaging of vulnerable atherosclerotic plaque
Authors: Tong, Wei and Hui, Hui and Shang, Wenting and Zhang, Yingqian and Tian, Feng and Ma, Qiang and Yang, Xin and Tian, Jie and Chen, Yundai
Journal: (2021)

Sight and switch off: Nerve density visualization for interventions targeting nerves in prostate cancer
Authors: You, Huijuan and Shang, Wenting and Min, Xiangde and Weinreb, Jeffrey and Li, Qiubai and Leapman, Michael and Wang, Liang and Tian, Jie
Journal: Science Advances (2020): eaax6040

Near-Infrared Dual-Emission Ratiometric Fluorescence Imaging Nanoprobe for Real-Time Tracing the Generation of Endogenous Peroxynitrite in Single Living Cells and In Vivo
Authors: Lin, Pengxiang and Chen, Dongxia and Zhang, Liangliang and Xu, Jiayao and Huang, Yong and Zhao, Shulin
Journal: ACS Omega (2020)

Hexapeptide-conjugated calcitonin for targeted therapy of osteoporosis
Authors: Liu, Yanpeng and Yu, Peng and Peng, Xu and Huang, Qin and Ding, Mingming and Chen, Yantao and Jin, Ruitao and Xie, Jing and Zhao, Changsheng and Li, Jianshu
Journal: Journal of Controlled Release (2019)

Activated platelets and antibody opsonization as tumor markers: novel avenues for cancer diagnosis, targeted therapy and monitoring therapeutic outcomes
Authors: Yap, May Lin
Journal: (2018)

Thermo-sensitive hydrogel PLGA-PEG-PLGA as a vaccine delivery system for intramuscular immunization
Authors: Wang, Xiaoyan and Zhang, Yu and Xue, Wei and Wang, Hong and Qiu, Xiaozhong and Liu, Zonghua
Journal: Journal of Biomaterials Applications (2017): 923--932

Cube-shaped theranostic paclitaxel prodrug nanocrystals with surface functionalization of SPC and MPEG-DSPE for imaging and chemotherapy
Authors: Guo, Fuqiang and Shang, Jiajia and Zhao, Hai and Lai, Kangrong and Li, Yang and Fan, Zhongxiong and Hou, Zhenqing and Su, Guanghao
Journal: Colloids and Surfaces B: Biointerfaces (2017)

Light/magnetic hyperthermia triggered drug released from multi-functional thermo-sensitive magnetoliposomes for precise cancer synergetic theranostics
Authors: Guo, Yuxin and Zhang, Yang and Ma, Jinyuan and Li, Qi and Li, Yang and Zhou, Xinyi and Zhao, Dan and Song, Hua and Chen, Qing and Zhu, Xuan
Journal: Journal of Controlled Release (2017)

Molecular Basis and Consequences of the Cytochrome c-tRNA Interaction
Authors: Liu, Cuiping and Stonestrom, Aaron J and Christian, Thomas and Yong, Jeongsik and Takase, Ryuichi and Hou, Ya-Ming and Yang, Xiaolu
Journal: Journal of Biological Chemistry (2016): 10426--10436

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

Quantitative proteomics by fluorescent labeling of cysteine residues using a set of two cyanine-based or three rhodamine-based dyes
Authors: Volke D, Hoffmann R.
Journal: Electrophoresis (2008): 4516

Surface plasmon resonance-enhanced fluorescence implementation of a single-step competition assay: demonstration of fatty acid measurement using an anti-fatty acid monoclonal antibody and a Cy5-labeled fatty acid
Authors: Vareiro MM, Tranchant I, Maplin S, Zak K, Gani MM, Slevin CJ, Hailes HC, Tabor AB, Cameron PJ, Jenkins AT, Williams DE.
Journal: Anal Biochem (2008): 243

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

Near-infrared fluorescence imaging of tumor integrin alpha v beta 3 expression with Cy7-labeled RGD multimers
Authors: Wu Y, Cai W, Chen X.
Journal: Mol Imaging Biol (2006): 226

Cy7-Bis-dipicolylamine-zinc
Authors: Leung K., undefined
Journal: In: Molecular Imaging and Contrast Agent Database (MICAD), Bethesda (MD). (2004)

Cy7-Tetrameric arginine-glycine-aspartic acid peptide
Authors: Cheng KT., undefined
Journal: In: Molecular Imaging and Contrast Agent Database (MICAD), Bethesda (MD). (2004)