Cyanine dyes
by Lumiprobe
http://www.lumiprobe.com/tech/cyanine-dyes
There are two varieties of cyanine dyes: non-sulfonated cyanines, and sulfonated cyanines. For many applications they are interchangeable, because their spectral properties are nearly identical. Both sulfonated and non-sulfonated dyes can be used for the labeling of biomolecules such as DNA and proteins. The difference between the dyes is their solubility: sulfo- dyes are water-soluble, and they do not use of organic co-solvent for the labeling in aqueous environment. They are less prone to aggregation in water. There are cases when one of the type of cyanines is desired (see Sulfonated vs non-sulfonated cyanines section below).
Non-sulfonated cyanines
Available non-sulfonated dyes incude Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5. Cy stands for 'cyanine', and first digit is number of carbon atoms between indolenine groups. Cy2 which is oxazole derivative rather than indolenin, is an exception from this rule. Suffix .5 is added for benzo-fused cyanines. Variation of the structures allows to change fluorescence properties of the molecules, and to cover most important part ot visible and NIR spectrum with several fluorophores.
Fluorescent properties of non-sulfonated cyanines have little dependence on solvent and surrounding. Absorbance and fluorescence spectra of non-sulfonated cyanine dyes are plotted below.
Sulfonated cyanines
Sulfonated cyanines include additional sulfo-groups which facilitate dissolution of dye molecules in aqueous phase. Charged sulfonate groups decrease aggregation of dye molecules and heavily labeled conjugates. Currently available sulfonated cyanines include sulfo-Cy3, sulfo-Cy5, and sulfo-Cy7.
Sulfonated vs non-sulfonated cyanines
Sulfonated and non-sulfonated cyanines exhibit very similar fluorescent properties. However, there are a few differences in labeling protocols that should be noticed. Non-sulfonated cyanines must be dissolved in organic co-solvent (DMF or DMSO) prior to use, and added to a solution of target molecule in aqueous buffers. Recommended volume of co-solvent should be 10% for Cy3, Cy5, Cy7, and 15% for .5 counterparts. Sulfo-Cy 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-Cy 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.
Sulfonated and non-sulfonated cyanines are interchangeable for the labeling of many classes of 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
Conjugates produced with similar sulfo- and non-sulfonated reagents (for example, sulfo-Cy5 and Cy5) are very similar in their fluorescent properties, and can be used with various fluorescence instrumentation.
Sulfonated cyanines must be used for:
- sensitive proteins which are denatured by DMF or DMSO
- protein conjugation when purification is done by dialysis
- nanoparticles in aqueous soluitons
- insoluble or hydrophobic proteins
Non-sulfonated cyanines must be used for:
- reactions in organic media (dichloromethane, acetonitrile)
read more at
http://www.lumiprobe.com/tech/cyanine-dyes
Nanotechnology and other Fluorescent citations:
http://www.lumiprobe.com/citations
Protocols
http://www.lumiprobe.com/protocols
Lumiprobe sponsors nanpaprika.eu
For 5% discount when you order use code: nanopap
Comments