Cyanine 5.5 monosuccinimidyl ester [equivalent to Cy5.5® 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	1317.70
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.05
Correction Factor (280 nm)	0.101
Correction Factor (482 nm)	0.0017
Correction Factor (565 nm)	0.047
Correction Factor (650 nm)	0.454
Extinction coefficient (cm ^-1 M ^-1)	250000
Excitation (nm)	683
Emission (nm)	703
Quantum yield	0.27

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

Molecular weight

1317.70

Correction Factor (260 nm)

0.05

Correction Factor (280 nm)

0.101

Correction Factor (482 nm)

0.0017

Correction Factor (565 nm)

0.047

Correction Factor (650 nm)

0.454

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

250000

Excitation (nm)

683

Emission (nm)

703

Quantum yield

0.27

A variety of cyanine 5.5 (Cy5.5®) 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. Cy5.5® dyes are one type of the most common red fluorophores. Cy5.5® 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. Cy5.5® 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 5.5 monosuccinimidyl ester stock solution (Solution B)

Add anhydrous DMSO into the vial of Cyanine 5.5 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 5.5 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 5.5 monosuccinimidyl ester [equivalent to Cy5.5® 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	75.89 µL	379.449 µL	758.898 µL	3.794 mL	7.589 mL
5 mM	15.178 µL	75.89 µL	151.78 µL	758.898 µL	1.518 mL
10 mM	7.589 µL	37.945 µL	75.89 µL	379.449 µL	758.898 µL

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)	0.101
Correction Factor (482 nm)	0.0017
Correction Factor (565 nm)	0.047
Correction Factor (650 nm)	0.454
Extinction coefficient (cm ^-1 M ^-1)	250000
Excitation (nm)	683
Emission (nm)	703
Quantum yield	0.27

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)	Correction Factor (650 nm)
Cyanine 3.5 monosuccinimidyl ester [equivalent to Cy3.5® NHS ester]	579	591	150000	0.15	0.08	0.178	-	-	-
Cyanine 5.5 bissuccinimidyl ester [equivalent to Cy5.5® bisNHS ester]	683	703	250000	0.27	0.05	0.101	0.0017	0.047	0.454

Images

Figure 1. Chemical structure for Cyanine 5.5 monosuccinimidyl ester [equivalent to Cy5.5® NHS ester]

Citations

View all 19 citations: Citation Explorer

Protocol to prepare doubly labeled fluorescent nucleosomes for single-molecule fluorescence microscopy
Authors: Ghoneim, Mohamed and Musselman, Catherine A
Journal: STAR protocols (2023): 102229

2-Methoxyestradiol Loaded Mesoporous Polydopamine Nanoprobes for Hypoxia Alleviation and Sorafenib Synergistic treatment of Hepatocellular Carcinoma
Authors: Wang, Peifeng and Du, Yang and Zhao, Xingyang and Hu, Yueyang and Zhou, Tianjun and Liang, Xiaolong and Fang, Chihua and Tian, Jie
Journal: Materials \& Design (2023): 112137

Design of Diselenide-Bridged Hyaluronic Acid Nano-antioxidant for Efficient ROS Scavenging to Relieve Colitis
Authors: Xu, Jiaqi and Chu, Tianjiao and Yu, Tingting and Li, Naishi and Wang, Chunling and Li, Chen and Zhang, Yinlong and Meng, Huan and Nie, Guangjun
Journal: ACS nano (2022): 13037--13048

Single-Molecule Characterization of Cy3. 5-Cy5. 5 Dye Pair for FRET Studies of Nucleic Acids and Nucleosomes
Authors: Ghoneim, Mohamed and Musselman, Catherine A
Journal: Journal of Fluorescence (2022): 1--9

A Hepatocellular Carcinoma Targeting Nanostrategy with Hypoxia-Ameliorating and Photothermal Abilities that, Combined with Immunotherapy, Inhibits Metastasis and Recurrence
Authors: Zhou, Tianjun and Liang, Xiaolong and Wang, Peifeng and Hu, Yueyang and Qi, Yafei and Jin, Yushen and Du, Yang and Fang, Chihua and Tian, Jie
Journal: ACS nano (2020): 12679--12696

Injectable reactive oxygen species-responsive SN38 prodrug scaffold with checkpoint inhibitors for combined chemoimmunotherapy
Authors: Gong, Yimou and Chen, Muchao and Tan, Yanjun and Shen, Jingjing and Jin, Qiutong and Deng, Wutong and Sun, Jian and Wang, Chao and Liu, Zhuang and Chen, Qian
Journal: ACS Applied Materials \& Interfaces (2020): 50248--50259

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 (2018): 145--158

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)

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
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Journal: Theranostics (2011): 381

Rational approach to select small peptide molecular probes labeled with fluorescent cyanine dyes for in vivo optical imaging
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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.
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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
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Thiazole orange and Cy3: improvement of fluorescent DNA probes with use of short range electron transfer
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Near-infrared fluorescence imaging of tumor integrin alpha v beta 3 expression with Cy7-labeled RGD multimers
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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
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