CF™647 Dye

CF™647 is a cyanine-based far-red fluorescent dye spectrally similar to Cy®5 and Alexa Fluor® 647 (Figure 1). Compared with the other dyes, CF™647 has good fluorescence brightness (Figures 2 and 3A). However, the most attractive feature of CF™647 is its superior signal-to-noise ratio for its antibody conjugates in immunostaining (Figure 3B). Alexa Fluor® 647 bears multiple negative charges, which improve the brightness of the dye but also significantly alter the isoelectric point of antibody conjugates, resulting in lowered specificity of the conjugates.

To overcome the problem, a polymer blocking agent, such as Image-it™, is recommended to pre-treat the sample prior to the staining. CF™647, on the other hand, is minimally charged. As a result, antibody conjugates prepared from the dye have excellent specificity.

A full selection of reactive dyes, secondary antibodies, antibody labeling kits, and other bioconjugates including phalloidins, Annexin V and α-bungarotoxin are available for CF™ dyes.

Figure 1. Absorption and emission spectra of CF™647 conjugated to goat anti-mouse IgG in PBS.

Figure 2. Jurkat cells were treated with staurosporine to induce apoptosis and stained with NucView™488 (live cell caspase-3 detection) and CF™647 Annexin-V. CF™647 Annexin-V staining is pseudocolored magenta.

Figure 3A. Jurkat cells were fixed, permeabilized and stained with intracellular mouse anti-human CD3 followed by CF™647 (Biotium), Alexa Fluor® 647 (Invitrogen) or other commercially available goat anti-mouse IgG conjugates. Stained cells were analyzed on a BD FACS Calibur in the FL4 channel. (A) The bars represent the relative geometric means of the cell population. Background was determined by secondary antibody staining alone. (B) The signal-to-noise ratio of the geometric means from A.

Figure 3B