Cyanine 3 monosuccinimidyl ester, potassium salt [same as GE Cy3® NHS ester]

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Additional ordering information

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

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)	150000¹
Excitation (nm)	555
Emission (nm)	569
Quantum yield	0.15¹

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

146368-16-3

Molecular weight

829.03

Correction Factor (260 nm)

0.07

Correction Factor (280 nm)

0.073

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

150000¹

Excitation (nm)

555

Emission (nm)

569

Quantum yield

0.15¹

This Cy3® dye is the same molecule to GE's Cy3® NHS ester. Cy3® NHS ester readily reacts with amino groups. A variety of cyanine 3 (Cy3®) 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. Cy3® dyes have enhanced fluorescence upon binding to proteins. Cy3® 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 3 monosuccinimidyl ester Potassium Salt stock solution (Solution B)

Add anhydrous DMSO into the vial of Cyanine 3 monosuccinimidyl ester Potassium Salt 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 3 monosuccinimidyl ester Potassium Salt. 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 3 monosuccinimidyl ester, potassium salt [same as GE 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
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Spectrum

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Spectral properties

Correction Factor (260 nm)	0.07
Correction Factor (280 nm)	0.073
Extinction coefficient (cm ^-1 M ^-1)	150000¹
Excitation (nm)	555
Emission (nm)	569
Quantum yield	0.15¹

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 bissuccinimidyl ester, potassium salt [same as GE Cy3® bisNHS ester]	555	569	150000¹	0.15¹	0.07	0.073	-	-
Cyanine 5 monosuccinimidyl ester, potassium salt [same as GE Cy5® NHS ester]	651	670	250000¹	0.27¹, 0.4²	0.02	0.03	0.009	0.09
Cyanine 7 monosuccinimidyl ester, potassium salt [same as GE Cy7® NHS ester]	756	779	250000	0.3	0.05	0.036	0.0005	0.0193

Images

Figure 1. HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin followed by AAT’s Cy3^® goat anti-mouse IgG conjugate (Red, Left) or Jackson’s goat anti-mouse IgG conjugated with Cy3^® (Red, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue, Cat# 17530).

Figure 2. Chemical structure for Cyanine 3 monosuccinimidyl ester, potassium salt [same as GE Cy3® NHS ester]

Citations

View all 21 citations: Citation Explorer

LLPS of FXR proteins drives replication organelle clustering for $\beta$-coronaviral proliferation
Authors: Li, Meng and Hou, Yali and Zhou, Yuzheng and Yang, Zhenni and Zhao, Hongyu and Jian, Tao and Yu, Qianxi and Zeng, Fuxing and Liu, Xiaotian and Zhang, Zheng and others,
Journal: Journal of Cell Biology (2024)

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,
Journal: (2024)

An ULK1/2-PXN mechanotransduction complex suppresses breast cancer cell migration
Authors: Liang, Peigang and Zhang, Jiaqi and Wu, Yuchen and Zheng, Shanyuan and Xu, Zhaopeng and Yang, Shuo and Wang, Jinfang and Ma, Suibin and Xiao, Li and Wu, Tianhui and others,
Journal: bioRxiv (2023): 2023--02

A liquid-to-solid phase transition of Cu/Zn superoxide dismutase 1 (SOD1) initiated by oxidation and disease mutation
Authors: Gu, Siyu and Xu, Ming and Chen, Long and Shi, Xiangyan and Luo, Shi-Zhong
Journal: Journal of Biological Chemistry (2022): 102857

Caveat fluorophore: an insiders’ guide to small-molecule fluorescent labels
Authors: Grimm, Jonathan B and Lavis, Luke D
Journal: Nature methods (2022): 149--158

Promotion of row 1--specific tip complex condensates by Gpsm2-G$\alpha$i provides insights into row identity of the tallest stereocilia
Authors: Shi, Yingdong and Lin, Lin and Wang, Chao and Zhu, Jinwei
Journal: Science Advances (2022): eabn4556

Promotion of row 1--specific tip complex condensates by Gpsm2-G i provides insights into row identity of the tallest stereocilia
Authors: Shi, Yingdong and Lin, Lin and Wang, Chao and Zhu, Jinwei
Journal: (2022)

Phase separation-mediated TARP/MAGUK complex condensation and AMPA receptor synaptic transmission
Authors: Zeng, Menglong and D{\'\i}az-Alonso, Javier and Ye, Fei and Chen, Xudong and Xu, Jia and Ji, Zeyang and Nicoll, Roger A and Zhang, Mingjie
Journal: Neuron (2019): 529--543

Myosin VII, USH1C, and ANKS4B or USH1G Together Form Condensed Molecular Assembly via Liquid-Liquid Phase Separation
Authors: He, Yunyun and Li, Jianchao and Zhang, Mingjie
Journal: Cell reports (2019): 974--986

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

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)