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Cyanine 3 monosuccinimidyl ester [equivalent to Cy3® NHS ester]

HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin followed by AAT&rsquo;s Cy3<sup>&reg;</sup>&nbsp;goat anti-mouse IgG conjugate (Red, Left) or Jackson&rsquo;s goat anti-mouse IgG conjugated with Cy3<sup>&reg;</sup>&nbsp; (Red, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue, Cat# 17530).
HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin followed by AAT&rsquo;s Cy3<sup>&reg;</sup>&nbsp;goat anti-mouse IgG conjugate (Red, Left) or Jackson&rsquo;s goat anti-mouse IgG conjugated with Cy3<sup>&reg;</sup>&nbsp; (Red, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue, Cat# 17530).
HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin followed by AAT&rsquo;s Cy3<sup>&reg;</sup>&nbsp;goat anti-mouse IgG conjugate (Red, Left) or Jackson&rsquo;s goat anti-mouse IgG conjugated with Cy3<sup>&reg;</sup>&nbsp; (Red, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue, Cat# 17530).
Gallery Image 2
H2O2 promotes SOD1 phase separation. Confocal microscopy images of SOD1(100 μM, 15% PEG) colocalized with 20 μM ThT in the presence or absence of 1 mM H2O2 (at room temperature for 24 h). Scale bar represents 10 μm. Source: Figure from <b>A liquid-to-solid phase transition of Cu/Zn superoxide dismutase 1 initiated by oxidation and disease mutation</b> by Gu et al. <em>Journal of Biological Chemistry</em>, Feb. 2023.
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Physical properties
Molecular weight829.03
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.07
Correction Factor (280 nm)0.073
Extinction coefficient (cm -1 M -1)1500001
Excitation (nm)555
Emission (nm)569
Quantum yield0.151
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501

OverviewpdfSDSpdfProtocol


CAS
146368-16-3
Molecular weight
829.03
Correction Factor (260 nm)
0.07
Correction Factor (280 nm)
0.073
Extinction coefficient (cm -1 M -1)
1500001
Excitation (nm)
555
Emission (nm)
569
Quantum yield
0.151
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
  1. 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.
  2. 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.
  1. Prepare Sephadex G-25 column according to the manufacture instruction.
  2. Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
  3. Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
  4. 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 mg0.5 mg1 mg5 mg10 mg
1 mM120.623 µL603.114 µL1.206 mL6.031 mL12.062 mL
5 mM24.125 µL120.623 µL241.246 µL1.206 mL2.412 mL
10 mM12.062 µL60.311 µL120.623 µL603.114 µL1.206 mL

Molarity calculator

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

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Spectrum


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spectrum

Spectral properties

Correction Factor (260 nm)0.07
Correction Factor (280 nm)0.073
Extinction coefficient (cm -1 M -1)1500001
Excitation (nm)555
Emission (nm)569
Quantum yield0.151

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)Correction Factor (482 nm)Correction Factor (565 nm)
Cyanine 3 bissuccinimidyl ester [equivalent to Cy3® bisNHS ester]55556915000010.1510.070.073--
Cyanine 5 monosuccinimidyl ester [equivalent to Cy5® NHS ester]65167025000010.271, 0.420.020.030.0090.09
Cyanine 7 monosuccinimidyl ester [equivalent to Cy7® NHS ester]7567792500000.30.050.0360.00050.0193

Images


Citations


View all 32 citations: Citation Explorer
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
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)
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
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
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
Arc weakens synapses by dispersing AMPA receptors from postsynaptic density via modulating PSD phase separation
Authors: Chen, Xudong and Jia, Bowen and Araki, Yoichi and Liu, Bian and Ye, Fei and Huganir, Richard and Zhang, Mingjie
Journal: Cell Research (2022): 914--930
Caveat fluorophore: an insiders’ guide to small-molecule fluorescent labels
Authors: Grimm, Jonathan B and Lavis, Luke D
Journal: Nature methods (2022): 149--158
Phase separation-mediated condensation of Whirlin-Myo15-Eps8 stereocilia tip complex
Authors: Lin, Lin and Shi, Yingdong and Wang, Mengli and Wang, Chao and Lu, Qing and Zhu, Jinwei and Zhang, Rongguang
Journal: Cell Reports (2021): 108770
A simple lectin-based biochip might display the potential clinical value of glycomics in patients with spontaneous intracerebral hemorrhage
Authors: Ye, Lei and Fang, Yong-Sheng and Li, Xiao-Xue and Gao, Yi and Liu, Sheng-Sheng and Chen, Qiang and Wu, Qiang and Cheng, Hong-Wei and Du, Wei-Dong
Journal: Annals of Translational Medicine (2021)
Kindlin2-mediated phase separation underlies integrin adhesion formation
Authors: Li, Yujie and Zhang, Ting and Li, Huadong and Yang, Haibin and Lin, Ruihong and Sun, Kang and Wang, Lei and Zhang, Jing and Wei, Zhiyi and Yu, Cong
Journal: BioRxiv (2020)

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
Surface plasmon resonance-enhanced fluorescence implementation of a single-step competition assay: demonstration of fatty acid measurement using an anti-fatty acid monoclonal antibody and a Cy5-labeled fatty acid
Authors: Vareiro MM, Tranchant I, Maplin S, Zak K, Gani MM, Slevin CJ, Hailes HC, Tabor AB, Cameron PJ, Jenkins AT, Williams DE.
Journal: Anal Biochem (2008): 243
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
Near-infrared fluorescence imaging of tumor integrin alpha v beta 3 expression with Cy7-labeled RGD multimers
Authors: Wu Y, Cai W, Chen X.
Journal: Mol Imaging Biol (2006): 226
Cy7-Bis-dipicolylamine-zinc
Authors: Leung K., undefined
Journal: In: Molecular Imaging and Contrast Agent Database (MICAD), Bethesda (MD). (2004)
Cy7-Tetrameric arginine-glycine-aspartic acid peptide
Authors: Cheng KT., undefined
Journal: In: Molecular Imaging and Contrast Agent Database (MICAD), Bethesda (MD). (2004)