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Cyanine dyes

Cyanine dyes are molecules containing polymethine bridges between two nitrogen atoms with a delocalizedcyanine_dyes charge which have been used for many years as dyes in life science. Since the Cy3, Cy5 and Cy7 were made commercially available as succinimidyl esters in the early 1990s, cyanine dyes began to be used widely as labels for nucleic acids. Because of the good properties of low non-specific binding to biomolecules and bright fluorescence owing to their huge extinction coefficients and good quantum yields, the Cy dyes have been widespread used in DNA and RNA labelling. Cyanine dyes can be divided into two groups based on water solubility: non-sulfonated cyanines and sulfonated cyanines.

Non-sulfonated cyanines
Available non-sulfonated dyes include Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5. Cy stands for 'cyanine', and the first digit identifies the number of carbon atoms between the indolenine groups. The suffix .5 is added for benzo-fused cyanines. Most derivatives of non-sulfonated cyanines have low aqueous solubility except for hydrochlorides of hydrazides and amines. They are organic co-solvent soluble (5-20% of DMF or DMSO). When these molecules are used for biomolecule labeling, they should be dissolved in organic solvent first, and added to a solution of biomolecule (protein, peptide, amino-labeled DNA) in appropriate aqueous buffer. Fluorescent properties of non-sulfonated cyanines have little dependence on solvent and surrounding.

Sulfonated cyanines
Available sulfonated cyanines include sulfo-Cy3, sulfo-Cy5, and sulfo-Cy7, which have additional sulfo-groups that facilitate dissolution of dye molecules in aqueous phase. Charged sulfonate groups decrease aggregation of dye molecules and heavily labeled conjugates. Sulfonated cyanines are highly water soluble, and they do not use organic co-solvent for the labeling in aqueous environment.

Application


Sulfonated and non-sulfonated cyanines exhibit very similar fluorescent properties. However, non-sulfonated cyanines must be dissolved in an organic co-solvent (DMF or DMSO) prior to use, and added to a solution of the target molecule in aqueous buffers. The recommended volume of co-solvent should be 10% for Cy3, Cy5, Cy7, and 15% for .5 counterparts. Sulfo-Cyanine reagents can be used in purely aqueous conditions. There is also a difference in purification: when dialysis against water or aqueous buffer is used for purification, sulfo-Cyanine must be used to achieve efficient removal of unreacted dye material. Reactions with both sulfo- and non-sulfo cyanines can be purified by gel filtration, chromatography (HPLC, FPLC, ion exchange), or electrophoresis.

Non-Sulfonated cyanines are available for the labeling of many targets including:
• Soluble proteins, which are tolerant to addition of organic co-solvent.
• Antibodies (use 5-10% of DMSO/DMF).
• DNA and oligonucleotides,
• Peptides.
• Many small molecules.
• Reactions in organic media (dichloromethane, acetonitrile).
Sulfonated cyanines are available for the labeling of many targets including:
• Soluble proteins, which are tolerant to addition of organic co-solvent.
• Antibodies (use 5-10% of DMSO/DMF).
• DNA and oligonucleotides.
• Peptides.
• Many small molecules.
• Sensitive proteins which are denatured by DMF or DMSO.
• Protein conjugation when purification is done by dialysis.
• Nanoparticles in aqueous solutions.
• Insoluble or hydrophobic proteins.

Biochemicals

  1. Cat.No. Product Name Information
  2. A8100 Cy3 NHS ester (non-sulfonated) Labeling of amino-groups in biomolecules.
  3. A8101 Cy3.5 NHS ester (non-sulfonated) Labeling of amino-groups in biomolecules.
  4. A8102 Cy5 NHS ester (non-sulfonated) Labeling of amino-groups in biomolecules.
  5. A8103 Cy5.5 NHS ester (non-sulfonated) Labeling of amino-groups in biomolecules.
  6. A8104 Cy7 NHS ester (non-sulfonated) Labeling of amino-groups in biomolecules.
  7. A8105 Cy7.5 NHS ester (non-sulfonated) Labeling of amino-groups in biomolecules.
  8. A8107 Cy3 NHS ester Labeling of amino-groups in biomolecules.
  9. A8108 Cy5 NHS ester Labeling of amino-groups in biomolecules.
  10. A8109 Cy7 NHS ester Labeling of amino-groups in biomolecules.
  11. A8111 Cy3 azide (non-sulfonated) used for Click chemistry to label alkyne-modified oligonucleotides
  12. A8112 Cy3.5 azide (non-sulfonated) used for Click chemistry to label alkyne-modified oligonucleotides
  13. A8113 Cy5 azide (non-sulfonated) used for Click chemistry to label alkyne-modified oligonucleotides
  14. A8114 Cy5.5 azide (non-sulfonated) used for Click chemistry to label alkyne-modified oligonucleotides
  15. A8115 Cy7 azide (non-sulfonated) used for Click chemistry to label alkyne-modified oligonucleotides
  16. A8116 Cy7.5 azide (non-sulfonated) used for Click chemistry to label alkyne-modified oligonucleotides
  17. A8127 Cy3 azide used for Click chemistry to label alkyne-modified oligonucleotides
  18. A8128 Cy5 azide used for Click chemistry to label alkyne-modified oligonucleotides
  19. A8130 Cy3 alkyne (non-sulfonated) used for Click chemistry to label any molecule bearing azide group.
  20. A8131 Cy5 alkyne (non-sulfonated) used for Click chemistry to label any molecule bearing azide group.
  21. A8132 Cy3 carboxylic acid (non-sulfonated) Non-reactive fluorophore, for control experiments, and for calibration
  22. A8133 Cy5 carboxylic acid (non-sulfonated) Non-reactive fluorophore, for control experiments, and for calibration
  23. A8134 Cy5.5 carboxylic acid (non-sulfonated) Non-reactive fluorophore, for control experiments, and for calibration
  24. A8135 Cy7 carboxylic acid (non-sulfonated) Non-reactive fluorophore, for control experiments, and for calibration
  25. A8136 Cy7.5 carboxylic acid (non-sulfonated) Non-reactive fluorophore, for control experiments, and for calibration
  26. A8137 Cy5 carboxylic acid Non-reactive fluorophore, for control experiments, and for calibration
  27. A8138 Cy3 maleimide (non-sulfonated) Labeling of cysteine residues, as well as other thiolated molecules
  28. A8264 Cy3.5 maleimide (non-sulfonated) Labeling of cysteine residues, as well as other thiolated molecules
  29. A8139 Cy5 maleimide (non-sulfonated) Labeling of cysteine residues, as well as other thiolated molecules
  30. A8140 Cy5.5 maleimide (non-sulfonated) Labeling of cysteine residues, as well as other thiolated molecules
  31. A8141 Cy7 maleimide (non-sulfonated) Labeling of cysteine residues, as well as other thiolated molecules
  32. A8142 Cy7.5 maleimide(non-sulfonated) Labeling of cysteine residues, as well as other thiolated molecules
  33. A8143 Cy5 amine (non-sulfonated) can be conjugated with reactive groups such as NHS esters, carboxy groups (after carbodiimide activation), and epoxides.
  34. A8144 Cy5 Boc-hydrazide (non-sulfonated) carbonyl-reactive dye
  35. A8145 Cy5 hydrazide (non-sulfonated) carbonyl-reactive dye
  36. A8261 Cy5.5 hydrazide (non-sulfonated) carbonyl-reactive dye
  37. A8265 Cy3 hydrazide (non-sulfonated) carbonyl-reactive dye
  38. A8266 Cy3.5 hydrazide (non-sulfonated) carbonyl-reactive dye

References

1.Flors C. DNA and chromatin imaging with super-resolution fluorescence microscopy based on single-molecule localization. Biopolymers. 2011 May;95(5):290-7.

2.Kricka LJ. Stains, labels and detection strategies for nucleic acids assays. Ann Clin Biochem. 2002 Mar;39(Pt 2):114-29.

3.Swatton JE, Prabakaran S, Karp NA, Lilley KS, Bahn S. Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE). Mol Psychiatry. 2004 Feb;9(2):128-43.

4.Kettenhofen NJ, Wang X, Gladwin MT, Hogg N. In-gel detection of S-nitrosated proteins using fluorescence methods. Methods Enzymol. 2008;441:53-71.