Cyanine3 NHS Ester
equivalent to Cy3® NHS ester
Cy3® NHS Ester (GE Healthcare) and Cyanine3 monosuccinimidyl ester are chemically equivalent, reactive dyes used for
fluorescence labeling biological molecules by targeting amino-groups.
Background
Cy3 is a yellow fluorescent dye belonging to the cyanine dyes family. It is sometimes also referred to as Sulfo-Cy3; the two are chemically equivalent. In contrast, non-sulfonated Cy3 does not have SO3- groups. Consequently, non-sulfonated Cy3 has lower water solubility than Cy3, as well as increased likelihood of forming aggregates in squeous solution. For this reason, sulfo-Cy3 and Cy3 are much more common than non-sulfonated Cy3.Usage
Cy3 NHS ester has a molecular weight of 829.03 daltons. It is supplied as a red powder and can be reconstituted using DMSO. The NHS ester functional group allows for easy conjugation to targets with primary amine groups, such as the lysine residue of proteins and antibodies or amine-modified oligonucleotides. Labeling reactions create a covalent bond between the dye and biomolecule; for the best efficiency, the reaction should occur in a buffer with pH = 8.5 ± 0.5. Once reacted, a degree of labeling (DOL) should be calculated for the conjugate. This is important as overlabeling (i.e. high DOL) can result in self-quenching of the Cy3 fluorohpore.Excitation / Emission
Cy3 has two important wavelengths: an excitation peak at 555 nm and an emission peak at 569 nm. When excited by a suitable light source, such as a 532 nm or 555 nm laser, it will emit yellow fluorescence. As such, compatible fluorescence channels include Cy3 or TRITC filter sets. Cy3 fluorescence is fairly resistant to changes in pH, tolerating pH 4 to pH 10.Applications
Cy3 can be conjugated to antibodies and used for immunofluorescence (IF) and immunocytochemistry (ICC) applications. For example, cells treated with mouse anti-tubulin primary antibodies, followed by Cy3 goat anti-mouse IgG secondary antibodies, can be used to visualize cytoskeletal structures.A second application is the use of Cy3 to label oligonucleotides. In fluorescence in situ hybridization (FISH), Cy3-oligo probes allow for spatial mapping of genetic material in cells. Combined with a quencher, Cy3-oligo conjugates can be used as TaqMan probes (or molecular beacons) for real-time PCR techniques.
Other formats
Cy3 is also available with maleimide moieties. This allows for conjugation to proteins and antibodies which present sulfhydryl-groups, such as cysteine residues. For incorporation of Cy3 into an oligonucleotide during chemical synthesis, Cy3 phosphoramidites can be used. In this process, phosphoramidites are added from the 3' to 5' direction.Cy5 is an alternative to Cy3 that belongs to the same family for fluorescent dyes. It has an excitation wavelength of 651 nm and an emission wavelength of 670 nm.
Cy3B is an analog of Cy3 that is more resistant to internal de-excitation, thus resulting in a measured increase in fluorescence signal and improved photostability.
iFluor 555 is a spectrally similar fluorophore that offers improved water solubility and brightness, with decreased photobleaching and cost.
Conjugation protocol
Quick summary
- Prepare protein stock solution
- Prepare dye stock solution
- Determine optimal dye/protein ratio (optional)
- Run conjugation reaction
- Purify the conjugation
Full protocol
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 | ||||
/ | = | 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 monosuccinimidyl ester [equivalent to Cy7® NHS ester] | 756 | 779 | 250000 | 0.3 | 0.05 | 0.036 | 0.0005 | 0.0193 |
Citations
View all 33 citations: Citation Explorer
CLASP-mediated competitive binding in protein condensates directs microtubule growth
Authors: 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 He, Sicong and others,
Journal: Nature Communications (2024): 6509
Authors: 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 He, Sicong and others,
Journal: Nature Communications (2024): 6509
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
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
Thiazole orange and Cy3: improvement of fluorescent DNA probes with use of short range electron transfer
Authors: Menacher F, Rubner M, Berndl S, Wagenknecht HA.
Journal: J Org Chem (2008): 4263
Authors: Menacher F, Rubner M, Berndl S, Wagenknecht HA.
Journal: J Org Chem (2008): 4263
Page updated on September 5, 2024