Cyanine 5.5 bissuccinimidyl ester, potassium salt [same as GE Cy5.5® bisNHS ester]

This Cy5.5® dye is the same molecule to GE's Cy5.5® bisNHS ester. It readily reacts with amino groups. It has two NHS groups that might give brighter signal on some proteins than the Cy5.5 mono NHS ester (#174 and #283). Our Cy5.5® Fluors are thoroughly QC tested to ensure high levels of chromophore and reactive dye content. 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 bissuccinimidyl ester Potassium Salt stock solution (Solution B)

Add anhydrous DMSO into the vial of Cyanine 5.5 bissuccinimidyl 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 5.5 bissuccinimidyl ester Postassium 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 5.5 bissuccinimidyl ester, potassium salt [same as GE Cy5.5® bisNHS 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	76.245 µL	381.225 µL	762.451 µL	3.812 mL	7.625 mL
5 mM	15.249 µL	76.245 µL	152.49 µL	762.451 µL	1.525 mL
10 mM	7.625 µL	38.123 µL	76.245 µL	381.225 µL	762.451 µL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

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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 5.5 monosuccinimidyl ester, potassium salt [same as Cy5.5® NHS ester]	683	703	250000	0.27	0.05	0.101	0.0017	0.047	0.454

Citations

View all 12 citations: Citation Explorer

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)

Carboxymethyl Dextran-Stabilized Polyethylenimine-Poly (epsilon-caprolactone) Nanoparticles-Mediated Modulation of MicroRNA-34a Expression via Small-Molecule Modulator for Hepatocellular Carcinoma Therapy
Authors: Deng, Xiongwei and Yin, Zhaoxia and Zhou, Zhixiang and Wang, Yihui and Zhang, Fang and Hu, Qin and Yang, Yishu and Lu, Jianqing and Wu, Yan and Sheng, Wang and others, undefined
Journal: ACS applied materials & interfaces (2016): 17068--17079

Affinity-Controlled Protein Encapsulation into Sub-30 nm Telodendrimer Nanocarriers by Multivalent and Synergistic Interactions
Authors: Wang, Xu and Shi, Changying and Zhang, Li and Bodman, Alexa and Guo, D and an , undefined and Wang, Lili and Hall, Walter A and Wilkens, Stephan and Luo, Juntao
Journal: Biomaterials (2016)

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

Page updated on December 17, 2024

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Physical properties

Molecular weight	1311.56
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