| Synonyms: | Equivalent of Cy5 |
| CAS #: | 146368-11-8 |
| Molecular Formula: | C33H40N2O8S2 |
| Molecular Weight: | 656.8 |
| Cy5, a member of the cyanine dye family, is a ubiquitous fluorescent molecule in biological and chemical research. Known for its distinct far-red emission, it has become a cornerstone of multi-color fluorescence experiments, enabling researchers to visualize cellular structures and molecular interactions with high specificity and low background noise. 1. Fundamental Properties and Spectral Characteristics Cy5 is a synthetic polymethine dye with an absorption and emission spectrum in the far-red region. This spectral location is its most significant advantage, as it falls outside the range where most biological samples exhibit autofluorescence. • Excitation/Emission Maxima: The dye typically has an excitation maximum (λex) around 649 nm and an emission maximum (λem) around 666−670 nm. This makes it an ideal partner for common laser lines, such as the 633 nm HeNe laser or the 647 nm diode laser. • igh Fluorescence Intensity: Cy5 is known for its high brightness, which is a key feature of cyanine dyes. It has a high extinction coefficient, meaning it absorbs light very efficiently, and a good quantum yield, which translates to a strong fluorescent signal. • Low Autofluorescence: The far-red emission is particularly useful because it minimizes interference from the natural fluorescence of biological materials like collagen, elastin, and flavins, which typically emit in the blue-green to orange spectrum. This results in an excellent signal-to-noise ratio, providing clearer images and more reliable data. • Photostability: While Cy5 is generally considered to have good photostability, it is less stable than some alternative dyes, such as the Alexa Fluor 647 dye. It is prone to photobleaching, which can limit its use in long-term, live-cell imaging or super-resolution microscopy experiments that require prolonged exposure to high-intensity light. 2. Common Applications in Biomedical Research Cy5’s unique spectral properties and high brightness have made it a go-to choice for a wide range of fluorescence-based techniques. • Fluorescence Microscopy: Cy5 is widely used for immunofluorescence (IF) and immunohistochemistry (IHC). When conjugated to a secondary antibody, it can be used to label specific proteins in cells or tissues, providing clear and detailed images of cellular localization and structure. Its far-red signal is perfect for multiplexing experiments, where it can be combined with blue and green dyes (e.g., DAPI and FITC) without significant spectral overlap. • Flow Cytometry: In flow cytometry, Cy5 is a popular fluorophore for analyzing cell populations. Its far-red emission can be detected by dedicated far-red channels, allowing for the simultaneous detection of multiple cellular markers without complex compensation or deconvolution. • Western Blotting: Cy5-labeled secondary antibodies are used for quantitative Western blotting. The use of fluorescent dyes like Cy5 offers several advantages over traditional chemiluminescent detection, including a wider dynamic range, better quantification, and the ability to detect multiple proteins on the same blot. • In Situ Hybridization (FISH): Cy5-labeled nucleic acid probes can be used to detect specific DNA or RNA sequences within cells and tissues, providing crucial information on gene expression and chromosomal abnormalities. • Super-Resolution Microscopy: Due to its photoswitchable properties, Cy5 is frequently used in super-resolution techniques like Stochastic Optical Reconstruction Microscopy (STORM). The dye can be reversibly switched between a fluorescent “on” state and a non-fluorescent “off” state, allowing for the precise localization of individual fluorophores and the creation of images with resolutions far beyond the diffraction limit. 3. Conjugation and Derivatives To be useful in biological applications, Cy5 must be chemically linked, or conjugated, to a biomolecule of interest (e.g., an antibody, protein, or oligonucleotide). It is typically supplied in a reactive form that allows for easy and efficient conjugation. 3.1 Reactive Groups: The most common reactive forms of Cy5 include: • N-hydroxysuccinimide (NHS) Ester: This is the most popular form for labeling primary amine groups (e.g., lysine residues on proteins or the 5′-amino group on oligonucleotides) under mild, basic conditions. • Maleimide: This derivative is used for conjugating Cy5 to thiol groups (e.g., cysteine residues), which allows for more specific labeling of proteins. • Click Chemistry Reagents: Cy5 can also be functionalized with azide or alkyne groups for “click chemistry” reactions, providing a highly specific and efficient way to label biomolecules. 3.2 Labeling Kits Many commercial kits are available that simplify the conjugation process, providing pre-measured reagents and streamlined protocols to quickly and efficiently label antibodies or other proteins. 4. Advantages and Disadvantages 4.1 Advantages • Far-Red Emission: Minimal biological autofluorescence, resulting in high signal-to-noise ratio. • High Brightness: Strong signal due to high extinction coefficient and good quantum yield. • Multiplexing Compatibility: Far-red spectrum allows for easy combination with other common fluorophores. • Versatility: Can be used in a wide range of applications, from microscopy to flow cytometry. 4.2 Disadvantages • Photobleaching: Less photostable than some modern alternatives (e.g., Alexa Fluor 647). • Dye Aggregation: Can self-quench at high degrees of labeling (high dye-to-protein ratios). • Environmental Sensitivity: The fluorescence of some cyanine dyes can be affected by the local environment (e.g., pH, polarity). • Requires Specific Equipment: Excitation and emission require far-red-compatible lasers and filters. 5. Cy5 vs. Alternatives While Cy5 is a workhorse dye, it’s important to consider its performance relative to other far-red fluorophores. The most common alternative is Alexa Fluor 647. In many head-to-head comparisons, Alexa Fluor 647 is found to be more photostable and less prone to self-quenching at high degrees of labeling, making it a better choice for demanding applications. However, Cy5’s ubiquity and lower cost often make it the preferred choice for routine experiments and as a standard in many established protocols. Cy5 fluorescent dyes remain an essential tool in the life sciences. Its distinctive far-red emission, high brightness, and compatibility with standard laboratory equipment make it an excellent choice for a wide array of applications, particularly in multi-color imaging and flow cytometry. While newer dyes offer improvements in photostability and brightness, Cy5’s reliability and widespread use ensure it will continue to be a staple in research for years to come. References: 1. Overview of Fluorescent Dyes 2. Cyanine 3. Dye Directory 4. Cy5 Dye Profile |
|
Cyanine 5
For Research & Development use only. Not for testing and/or use on humans.
