Cyanine 3 maleimide [equivalent to Cy3® maleimide]

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	866.92
Solvent	DMSO

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 yield	0.15¹

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501

Direct upgrades

Alternative formats

Cyanine 3 amine [equivalent to Cy3® amine]

Cyanine 3 azide [equivalent to Cy3® azide]

Cyanine 3 alkyne [equivalent to Cy3® alkyne]

Cyanine 3 hydrazide [equivalent to Cy3® hydrazide]

Overview SDS Protocol

Upgrade: iFluor® 555 maleimide

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. Cyanine 3 maleimide stock solution (Solution B)

Add anhydrous DMSO into the vial of Cyanine 3 maleimide 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 upto 4 weeks when kept from light and moisture. Avoid freeze-thaw cycles.

2. Protein stock solution (Solution A)

Mix 100 µL of a reaction buffer (e.g., 100 mM MES buffer with pH ~6.0) 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 6.5 ± 0.5. Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or other proteins will not be labeled well. 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.

3. Optional

if your protein does not contain a free cysteine, you must treat your protein with DTT or TCEP to generate a thiol group. DTT or TCEP are used for converting a disulfide bond to two free thiol groups. If DTT is used you must remove free DTT by dialysis or gel filtration before conjugating a dye maleimide to your protein. Following is a sample protocol for generating a free thiol group:

Prepare a fresh solution of 1 M DTT (15.4 mg/100 µL) in distilled water.
Make IgG solution in 20 mM DTT: add 20 µL of DTT stock per ml of IgG solution while mixing. Let stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
Pass the reduced IgG over a filtration column pre-equilibrated with "Exchange Buffer". Collect 0.25 mL fractions off the column.
Determine the protein concentrations and pool the fractions with the majority of the IgG. This can be done either spectrophotometrically or colorimetrically.
Carry out the conjugation as soon as possible after this step (see Sample Experiment Protocol). Note: IgG solutions should be >4 mg/mL for the best results. The antibody should be concentrated if less than 2 mg/mL. Include an extra 10% for losses on the buffer exchange column. Note: The reduction can be carried out in almost any buffers from pH 7-7.5, e.g., MES, phosphate or TRIS buffers. Note: Steps 3 and 4 can be replaced by dialysis.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with Cyanine 3 maleimide. 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 3 maleimide [equivalent to Cy3® maleimide] 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	115.351 µL	576.754 µL	1.154 mL	5.768 mL	11.535 mL
5 mM	23.07 µL	115.351 µL	230.702 µL	1.154 mL	2.307 mL
10 mM	11.535 µL	57.675 µL	115.351 µL	576.754 µL	1.154 mL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

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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 yield	0.15¹

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
Cyanine 3 bisacid [equivalent to Cy3® bisacid]	555	569	150000¹	0.15¹	0.07	0.073
Cyanine 3 monoacid [equivalent to Cy3® acid]	555	569	150000¹	0.15¹	0.07	0.073
Cyanine 5 maleimide [equivalent to Cy5® maleimide]	651	670	250000¹	0.27¹, 0.4²	0.02	0.03
Cyanine 7 maleimide [equivalent to Cy7® maleimide]	756	779	250000	0.3	0.05	0.036
Cyanine-3- dUTP [Cy3-dUTP] 1 mM in Tris Buffer (pH 7.5)	555	569	150000¹	0.15¹	0.07	0.073

Images

Figure 1. Chemical structure for Cyanine 3 maleimide [equivalent to Cy3® maleimide]

Citations

View all 17 citations: Citation Explorer

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)

Conformational compaction as a mechanism for ATP resolubilization of protein condensates
Authors: Gong, Zhou and Zhu, Yueling and Lin, Shiyan and Meng, Ling-Shen and Sun, Min and Liu, Maili and Li, Jingyuan and Tang, Chun
Journal: (2023)

Caveat fluorophore: an insiders’ guide to small-molecule fluorescent labels
Authors: Grimm, Jonathan B and Lavis, Luke D
Journal: Nature methods (2022): 149--158

Spatiotemporal stop-and-go dynamics of the mitochondrial TOM core complex correlates with channel activity
Authors: Wang, Shuo and Findeisen, Lukas and Leptihn, Sebastian and Wallace, Mark I and H{\"o}rning, Marcel and Nussberger, Stephan
Journal: Communications biology (2022): 1--11

Spatiotemporal Stop-and-go Dynamics of the Mitochondrial TOM Core Complex Correlates With Three-state Channel Activity
Authors: Wang, Shuo and Findeisen, Lukas and Leptihn, Sebastian and Wallace, Mark and H{\"o}rning, Marcel and Nussberger, Stephan
Journal: (2021)

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

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)

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)

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

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

References

View all 21 references: Citation Explorer

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Theranostic cRGD-BioShuttle Constructs Containing Temozolomide- and Cy7 For NIR-Imaging and Therapy
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Rational approach to select small peptide molecular probes labeled with fluorescent cyanine dyes for in vivo optical imaging
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In vivo detection of embryonic stem cell-derived cardiovascular progenitor cells using Cy3-labeled Gadofluorine M in murine myocardium
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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
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Near-infrared fluorescence imaging of tumor integrin alpha v beta 3 expression with Cy7-labeled RGD multimers
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Cy7-Bis-dipicolylamine-zinc
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