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Cyanine 5 bissuccinimidyl ester [equivalent to Cy5® bisNHS ester]
A variety of cyanine 5 ( Cy5®) 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® dyes are one type of the most common red fluorophores. These versatile fluorophores can tolerate a pH range of 3-10 for use in a variety of applications at biologically relevant pHs. The dyes are also DMSO tolerant and photostable to enable transfer from storage to assay without loss of performance. The aqueous solubility eliminates the need for organic solvents in the assay buffers. Our Cy5® Fluors are thoroughly QC tested to ensure high levels of chromophore and reactive dye content. Mono-reactive dyes are suitable for targeted, precise labeling of proteins and oligonucleotides and bis-reactive dyes are more suitable for general labeling. NHS ester dyes are recommended for labeling amine groups and maleimide dyes are recommended for labeling thiol groups. This Cy5® 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® 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 bissuccinimidyl ester stock solution (Solution B)
Add anhydrous DMSO into the vial of Cyanine 5 bissuccinimidyl 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 bissuccinimidyl 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 bissuccinimidyl ester [equivalent to Cy5® 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 | 96.318 µL | 481.589 µL | 963.178 µL | 4.816 mL | 9.632 mL |
| 5 mM | 19.264 µL | 96.318 µL | 192.636 µL | 963.178 µL | 1.926 mL |
| 10 mM | 9.632 µL | 48.159 µL | 96.318 µL | 481.589 µL | 963.178 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
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 bissuccinimidyl ester [equivalent to Cy7® bisNHS ester] | 756 | 779 | 250000 | 0.3 | 0.05 | 0.036 | 0.0005 | 0.0193 |
Citations
View all 13 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
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)
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)
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)
Molecular Basis and Consequences of the Cytochrome c-tRNA Interaction
Authors: Liu, Cuiping and Stonestrom, Aaron J and Christian, Thomas and Yong, Jeongsik and Takase, Ryuichi and Hou, Ya-Ming and Yang, Xiaolu
Journal: Journal of Biological Chemistry (2016): 10426--10436
Authors: Liu, Cuiping and Stonestrom, Aaron J and Christian, Thomas and Yong, Jeongsik and Takase, Ryuichi and Hou, Ya-Ming and Yang, Xiaolu
Journal: Journal of Biological Chemistry (2016): 10426--10436
Click-electron microscopy for imaging metabolically tagged nonprotein biomolecules
Authors: Ngo, John T and Adams, Stephen R and Deerinck, Thomas J and Boassa, Daniela and Rodriguez-Rivera, Frances and Palida, Sakina F and Bertozzi, Carolyn R and Ellisman, Mark H and Tsien, Roger Y
Journal: Nat Chem Biol (2016): 459--465
Authors: Ngo, John T and Adams, Stephen R and Deerinck, Thomas J and Boassa, Daniela and Rodriguez-Rivera, Frances and Palida, Sakina F and Bertozzi, Carolyn R and Ellisman, Mark H and Tsien, Roger Y
Journal: Nat Chem Biol (2016): 459--465
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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