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Cyanine 3 monosuccinimidyl ester [equivalent to Cy3® NHS ester]
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Application notes
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® 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. 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 stock solution (Solution B)
Add anhydrous DMSO into the vial of Cyanine 3 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 3 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 3 monosuccinimidyl ester [equivalent to 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
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 [equivalent to Cy3® bisNHS 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 monosuccinimidyl ester [equivalent to Cy7® NHS ester] | 756 | 779 | 250000 | 0.3 | 0.05 | 0.036 | 0.0005 | 0.0193 |
Citations
View all 32 citations: Citation Explorer
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)
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)
The disordered protein SERF promotes $\alpha$-Synuclein aggregation through liquid-liquid phase separation
Authors: Liu, He-Ning and Wang, Ting and Hu, Jin-Jian and Chen, Long and Shi, Xiangyan and Li, Yan-Mei and Luo, Shi-Zhong
Journal: Journal of Biological Chemistry (2024): 105667
Authors: Liu, He-Ning and Wang, Ting and Hu, Jin-Jian and Chen, Long and Shi, Xiangyan and Li, Yan-Mei and Luo, Shi-Zhong
Journal: Journal of Biological Chemistry (2024): 105667
Manganese regulation of COPII condensation controls circulating lipid homeostasis
Authors: Wang, Xiao and Huang, Runze and Wang, Yawei and Zhou, Wenjing and Hu, Yating and Yao, Yuanhang and Cheng, Kunlun and Li, Xin and Xu, Bolin and Zhang, Jie and others,
Journal: Nature Cell Biology (2023): 1--14
Authors: Wang, Xiao and Huang, Runze and Wang, Yawei and Zhou, Wenjing and Hu, Yating and Yao, Yuanhang and Cheng, Kunlun and Li, Xin and Xu, Bolin and Zhang, Jie and others,
Journal: Nature Cell Biology (2023): 1--14
Temporal and spatial assembly of inner ear hair cell ankle link condensate through phase separation
Authors: Wang, Huang and Du, Haibo and Ren, Rui and Du, Tingting and Lin, Lin and Feng, Zhe and Zhao, Dange and Wei, Xiaoxi and Zhai, Xiaoyan and Wang, Hongyang and others,
Journal: Nature Communications (2023): 1657
Authors: Wang, Huang and Du, Haibo and Ren, Rui and Du, Tingting and Lin, Lin and Feng, Zhe and Zhao, Dange and Wei, Xiaoxi and Zhai, Xiaoyan and Wang, Hongyang and others,
Journal: Nature Communications (2023): 1657
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
Authors: Gu, Siyu and Xu, Ming and Chen, Long and Shi, Xiangyan and Luo, Shi-Zhong
Journal: Journal of Biological Chemistry (2022): 102857
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
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
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
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
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
Authors: Volke D, Hoffmann R.
Journal: Electrophoresis (2008): 4516