Protocol for: Detection Sensitivity of Carestream Image Station 4000MM PRO for Three Commonly Used Fluorescent Dyes of the CyDye Family (Cy3, Cy5 and Cy7)
Abstract or Description:
Fluorescent dyes with longer wavelength emission maxima in the visible and near infrared ranges have become valuable tools in Western blotting applications. In particular the sulfonated indocyanine fluorescent dyes, CyDyesTM, (GE Healthcare, Piscataway, N.J., USA) are a single family of small bright fluorescent molecules that are widely used for labeling of biomolecules including antibodies and other proteins1,2. These molecules all derive from a common core structure (see Figure 1, top). Among their desirable properties are good aqueous solubility and relative insensitivity to pH changes between 3 and 10. They exhibit low non-specific binding permitting reliable activity in a wide range of buffers. CyDyes also have good photostability, high molar extinction coefficients and favorable quantum yields3. They are available in a variety of chemistries making them suitable for labeling via amine, thiol, and aldehyde groups4,5,6.
The purpose of the following study was to determine the detection sensitivity of the Carestream Image Station 4000MM PRO (IS4000MM PRO) with 10 X zoom lens for three commonly used members of the CyDye family (Cy3, Cy5 and Cy7) and to demonstrate their usefulness in multiplex Western blotting experiments. The 3 dyes have non-overlapping emission spectra (see Figure 1, bottom) making them ideal for the design of multiplexing protocols. They are routinely conjugated to IgG antibodies with approximately 6 to 9 moles of dye/antibody ratios. While specific labeling information is not available from all suppliers, higher ratios have been reported by Berlier and co-workers1 that result in self-quenching and diminished signal output. Quantum yields for these three forms of the fluorochromes have been estimated as > 0.15 for Cy3, > 0.28 for Cy5, and > 0.25 for Cy7.
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