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AAT Bioquest

Cyanine 5 monosuccinimidyl ester [equivalent to Cy5® NHS ester]

HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin followed by AAT&rsquo;s Cy5<sup>&reg;</sup>&nbsp;goat anti-mouse IgG conjugate (Red, Left) or Jackson&rsquo;s Cy5<sup>&reg;</sup>&nbsp;goat anti-mouse IgG conjugate (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 Cy5<sup>&reg;</sup>&nbsp;goat anti-mouse IgG conjugate (Red, Left) or Jackson&rsquo;s Cy5<sup>&reg;</sup>&nbsp;goat anti-mouse IgG conjugate (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 Cy5<sup>&reg;</sup>&nbsp;goat anti-mouse IgG conjugate (Red, Left) or Jackson&rsquo;s Cy5<sup>&reg;</sup>&nbsp;goat anti-mouse IgG conjugate (Red, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue, Cat# 17530).
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Telephone1-800-990-8053
Fax1-800-609-2943
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Physical properties
Molecular weight855.07
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.02
Correction Factor (280 nm)0.03
Correction Factor (482 nm)0.009
Correction Factor (565 nm)0.09
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)651
Emission (nm)670
Quantum yield0.271, 0.42
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
146368-14-1
Molecular weight
855.07
Correction Factor (260 nm)
0.02
Correction Factor (280 nm)
0.03
Correction Factor (482 nm)
0.009
Correction Factor (565 nm)
0.09
Extinction coefficient (cm -1 M -1)
2500001
Excitation (nm)
651
Emission (nm)
670
Quantum yield
0.271, 0.42
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 monosuccinimidyl ester stock solution (Solution B)
Add anhydrous DMSO into the vial of Cyanine 5 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 5 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 5 monosuccinimidyl ester [equivalent to Cy5® 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 mM116.949 µL584.747 µL1.169 mL5.847 mL11.695 mL
5 mM23.39 µL116.949 µL233.899 µL1.169 mL2.339 mL
10 mM11.695 µL58.475 µL116.949 µL584.747 µL1.169 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)0.02
Correction Factor (280 nm)0.03
Correction Factor (482 nm)0.009
Correction Factor (565 nm)0.09
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)651
Emission (nm)670
Quantum yield0.271, 0.42

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 monosuccinimidyl ester [equivalent to Cy3® NHS ester]55556915000010.1510.070.073--
Cyanine 5 bissuccinimidyl ester [equivalent to Cy5® bisNHS 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 25 citations: Citation Explorer
Structural basis of ELKS/Rab6B interaction and its role in vesicle capturing enhanced by liquid-liquid phase separation
Authors: Jin, Gaowei and Lin, Leishu and Li, Kaiyue and Li, Jiashan and Yu, Cong and Wei, Zhiyi
Journal: Journal of Biological Chemistry (2023): 104808
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
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
Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization
Authors: Xiao, Lu and Liao, Renjie and Guo, Jia
Journal: Molecules (2020): 4900
A novel matrix protein PfX regulates shell ultrastructure by binding to specific calcium carbonate crystal faces
Authors: Yang, Xue and Yang, Dong and Yan, Yi and Li, Shiguo and Han, Zaiming and Ji, Yinghui and Zheng, Guilan and Xie, Liping and Zhang, Rongqing
Journal: International Journal of Biological Macromolecules (2020)
A basic protein, N25, from a mollusk modifies calcium carbonate morphology and shell biomineralization
Authors: Yang, Dong and Yan, Yi and Yang, Xue and Liu, Jun and Zheng, Guilan and Xie, Liping and Zhang, Rongqing
Journal: Journal of Biological Chemistry (2019): jbc--RA118
Highly Multiplexed Single Cell In Situ Transcriptomic Analysis.
Authors: Xiao, Lu
Journal: (2019)
Myosin VII, USH1C, and ANKS4B or USH1G Together Form Condensed Molecular Assembly via Liquid-Liquid Phase Separation
Authors: He, Yunyun and Li, Jianchao and Zhang, Mingjie
Journal: Cell reports (2019): 974--986
Single-cell in situ RNA analysis with switchable fluorescent oligonucleotides
Authors: Xiao, Lu and Guo, Jia
Journal: Frontiers in cell and developmental biology (2018): 42
Structurally modulated codelivery of siRNA and Argonaute 2 for enhanced RNA interference
Authors: Li, Jiahe and Wu, Connie and Wang, Wade and He, Yanpu and Elkayam, Elad and Joshua-Tor, Leemor and Hammond, Paula T
Journal: Proceedings of the National Academy of Sciences (2018): E2696--E2705

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)