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Cyanine 5 maleimide [equivalent to Cy5® maleimide]

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Physical properties
Molecular weight892.96
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


See also: Cyanines
Molecular weight
892.96
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® maleimide readily reacts with thiol groups. 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. Cyanine 5 maleimide stock solution (Solution B)
Add anhydrous DMSO into the vial of Cyanine 5 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:
  1. Prepare a fresh solution of 1 M DTT (15.4 mg/100 µL) in distilled water.
  2. 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).
  3. Pass the reduced IgG over a filtration column pre-equilibrated with "Exchange Buffer". Collect 0.25 mL fractions off the column.
  4. Determine the protein concentrations and pool the fractions with the majority of the IgG. This can be done either spectrophotometrically or colorimetrically.
  5. 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 5 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
  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 maleimide [equivalent to Cy5® 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 mg0.5 mg1 mg5 mg10 mg
1 mM111.987 µL559.935 µL1.12 mL5.599 mL11.199 mL
5 mM22.397 µL111.987 µL223.974 µL1.12 mL2.24 mL
10 mM11.199 µL55.994 µL111.987 µL559.935 µL1.12 mL

Molarity calculator

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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)
Fluorescein-5-maleimide *CAS 75350-46-8*4985178000010.79001, 0.9520.320.35--
Cyanine 3 maleimide [equivalent to Cy3® maleimide]55556915000010.1510.070.073--
Cyanine 5 monoacid [equivalent to Cy5® acid]65167025000010.271, 0.420.020.030.0090.09
Cyanine 7 maleimide [equivalent to Cy7® maleimide]7567792500000.30.050.0360.00050.0193
5-TAMRA Maleimide [Tetramethylrhodamine-5-maleimide] *CAS 154480-30-5*55257890000-0.320.178--
Cyanine 5 bisacid [equivalent to Cy5® bisacid]65167025000010.271, 0.420.020.030.0090.09
Cyanine-5- dUTP [Cy5-dUTP] *1 mM in Tris Buffer (pH 7.5)*65167025000010.271, 0.420.020.030.0090.09
Cyanine 5-dATP [Cy5-dATP]65167025000010.271, 0.420.020.030.0090.09

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Citations


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References


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Theranostic cRGD-BioShuttle Constructs Containing Temozolomide- and Cy7 For NIR-Imaging and Therapy
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