Alexa Fluor® Dyes—Brightest Conjugates
Alexa Fluor® dye molecules can be attached to proteins without significant self-quenching, leading to brighter conjugates. Whether it is Alexa Fluor® 488 (Figure 1), Alexa Fluor® 555 (Figure 2), or Alexa Fluor® 647 (Figure 3), or any other Alexa Fluor® dye, you will get the brightest conjugate available for imaging, flow cytometry, and other fluorescence-based applications.
Figure 1. Comparison of relative fluorescence of goat anti–mouse IgG antibody conjugates prepared from the Alexa Fluor® 488 dye and fluorescein isothiocyanate (FITC). Conjugate fluorescence is measured by comparing the fluorescence quantum yield of the conjugated dye relative to that of a reference dye and multiplying by the dye:protein labeling ratio.
Figure 2. Comparison of relative fluorescence of goat anti–rabbit IgG antibody conjugates of the Alexa Fluor® 555 and Cy®3 dyes (prepared by Invitrogen) at different dye:protein ratios in the conjugate.
Figure 3. Flow cytometry comparison of the brightness of Alexa Fluor® 647 goat anti–mouse IgG antibody (red, A21235) with Cy5 goat anti–mouse IgG antibody from Jackson ImmunoResearch Laboratories (green) and Amersham-Pharmacia Biotech (blue). Human blood was blocked with normal goat serum and incubated with an anti-CD3 mouse monoclonal antibody; cells were washed, resuspended and incubated with either an Alexa Fluor® 647 or Cy5 goat anti–mouse IgG secondary antibody. Samples were analyzed on a flow cytometer equipped with a 633 nm He–Ne laser and a longpass emission filter (>650 nm).
Use Antifade Reagents for Improved Signal Stability
Whether using Alexa Fluor® dyes or other standard dyes, you can suppress photobleaching and preserve signal brightness by using Prolong® Gold Antifade. Compatible with a multitude of dyes across the spectrum, this is a valuable tool for multicolor applications.
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