Cy5 Fluorescent Conjugates
Cy Dyes offer intense fluorescence when coupled KPL’s affinity purified antibodies and streptavidin. The cyanine dye is a water-soluble, orange fluorescing compound that produces an intense signal. The sensitivity and reproducibility of KPL antibodies combined with the brightness of Cy5 dyes produce an exceptional set of conjugates ideal for multiple labeling experiments. Dye/protein ratios have been established to ensure optimal fluorescence with minimal background.
Confocal fluorescence micrograph of HeLa cells stained with monoclonal antibody against mitochondria enzyme and Cy3-conjugated anti-mouse antibody (red); rabbit polyclonal antibody to histones in DNA and Cy5-conjugated anti-rabbit antibody (blue).
Our extensive line of CyDye conjugates is available across a range of animal species, including human, mouse, rabbit and rat antibodies, as well as other animal species and streptavidin. They are affinity purified and in some cases further adsorbed to minimize cross-reactivity between animal species or shared reactivity with other immunoglobulin classes. KPL mouse antibodies are cross-adsorbed against human serum to minimize cross-reactivity. Anti-heavy and light chain antibodies provide species-specific detection of the IgG class. Antibodies directed against heavy chains react specifically with the heavy chain in the product name; light chain reactivity has been removed. F(ab’)2 fragment antibodies are also available for assays requiring extremely low background levels and absence of F(c)-mediated binding.
Cyanine dyes are excellent alternatives to many other fluorescein dyes as they are brighter and offer greater photostability. Cy5 conjugates are excited maximally at 650 nm and fluoresce maximally at 670 nm. They are excited to about 98% of maximum with a krypton/argon laser (647 nm line) or to about 63% of maximum with a helium/neon laser (633 nm line). Cy5 produces minimal autofluorescence of biological specimens in this region of the spectrum.
Cy3 and Cy5 Conjugates are Ideal for Multiple Labeling
KPL also offers Cy3 conjugates which are excited maximally at 550 nm and emit maximally at 570 nm in the red end of the spectrum. Instrumentation is able to easily distinguish between the two signals when one antigen is labeled with Cy3 and the other with Cy5. A confocal microscope equipped with the appropriate laser for excitation and a far-red detector enables double labeling with Cy3 and Cy5.
Microplates were coated with serially diluted mouse IgG at the indicated concentration. Conjugates at a concentration of 0.01 mg/mL were applied and incubated for 30 minutes. Fluorescence was measured with a Perkin Elmer VICTOR 3 Multilabel Plate Reader.