CyDye Fluors for Protein Arrays

Introduction CyDyes for protein arrays Properties and performance benefits of CyDye fluors Protein array results with Cy3 and Cy5 Spectral characteristics of CyDye fluors Introduction CyDye is the brand name for the Amersham Biosciences range of fluorescent dyes. CyDye fluors form the core of many key technologies and platforms. As highly sensitive molecular labeling tools, CyDye fluors are used in fields as diverse as DNA microarrays, 2-D protein electrophoresis, DNA sequencing, high-throughput screening and flow cytometry. Protein array technology is the latest application area to exploit the properties of CyDye fluors, which are particularly suited to highly sensitive and accurate differential analysis. CyDye fluors offer bright and intense colors with narrow emission bands and are spectrally distinct, making them exceptional dyes for multicolor detection in many applications. CyDyes for protein arrays With the introduction of two new products, Cy3 and Cy5 monoreactive dyes: protein array grade, CyDye fluors are now available in a format designed to meet the needs of the protein array researcher. Eack kit contains two vials of dye - sufficient for 20 to 80 slides, depending on sample. Ordering information Cy3 Mono-Reactive Dye Protein Array Grade Reagents to label proteins for protein arrays with Cy3 mono functional dye. Two securitainers each containing sufficient dried dye to label 1 mg protein. Sufficient for 20 to 80 slides, depending on sample.

Cy5 Mono-Reactive Dye Protein Array Grade Reagents to label proteins for protein arrays with Cy5 mono functional dye. Two securitainers each containing sufficient dried dye to label 1 mg protein. Sufficient for 20 to 80 slides, depending on sample.

N.B. CyDyes should be stored refrigerated at 2 - 8 °C in the dark. Properties and performance benefits of CyDye fluors Bright, intense dyes—very high extinction coefficients and favorable quantum yields means sensitive detection limits. CyDye fluors give bright signals thus increasing the amount of information that can be gained from protein array analyses. High sensitivity and dynamic range—meaningful data can be generated from low-abundance proteins. A linear signal of greater than four orders of magnitude can be obtained. Spectrally resolved dyes—for multiplexing, giving discreet, discernible signals. The CyDye absorption and emission spectra are sharp, resulting in good spectral separation of the different fluors. pH insensitive between 3–10—all CyDye fluors can be used at biologically relevant pH. .. Robust—survive harsh experimental conditions (e.g. urea, detergent). Good aqueous solubility—assays can be run in near in vivo conditions, no need for organic solvents. Purity—quality tested to ensure high levels of chromophore and reactive dye content. Superior photostability compared to other fluorescent dyes such as fluorescein. Protein array results with Cy3 and Cy5 Labeling with CyDye fluors gives high sensitivity detection of labeled proteins in a protein array. The result of a protein array obtained using Cy™3 and Cy5 CyDye fluors and proprietary slide chemistry is shown below. Differential analysis of sheep serum samples A and B, spiked with different cytokine concentrations, was performed on protein arrays. The array comprised 39 antibodies and included positive and negative controls, as well as Cy3/Cy5-labeled BSA standards. Cy3 fluorescence is represented in green and Cy5 fluorescence in red. Sample A was labeled with Cy3 and sample B was labeled with Cy5. The samples were combined and used to probe the array. The separate Cy3 and Cy5 images and the overlaid images are shown on the right. The Cy3/Cy5 overlay shows that the combined sample contains higher levels of Cy5 IL1 and IL1 relative to Cy3 IL1 and IL1 and lower levels of Cy5 IL6 relative to Cy3 IL6. The observed differences were generally in close agreement with the expected values. The differences were reproducible as demonstrated in the reverse experiment (data not shown). .. Spectral characteristics of CyDye fluors Fluorescence can be simply defined as the molecular absorption of light energy (photons) at one wavelength and its re-emission at another, usually longer wavelength. Technical specifications as well as excitation/emission spectra of Cy3 and Cy5 fluors are shown below. Fluorophore Color of fluorescence Absorption max. (nm) Fluorescence max. (nm) Extinc. coeff. (M-1cm-1) Molecular weight (Mr), Mono- functional Cy3 Orange 550 570 150 000 765.95 Cy5 Red 649 670 250 000 791.99