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Cyanine 3 monosuccinimidyl ester, potassium salt [same as GE Cy3® NHS ester]
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.
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, 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 |
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)
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)
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
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
Authors: Gu, Siyu and Xu, Ming and Chen, Long and Shi, Xiangyan and Luo, Shi-Zhong
Journal: Journal of Biological Chemistry (2022): 102857
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
Authors: Shi, Yingdong and Lin, Lin and Wang, Chao and Zhu, Jinwei
Journal: Science Advances (2022): eabn4556
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
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