RRX (Rhodamine Red-X) conjugates have a peak of excitation at 570 nm and a peak of emission at 590 nm. Although TRITC has been used traditionally with FITC for double labeling, better color separation is achieved by using RRX or Alexa Fluor® 594. Rhodamine Red-X is particularly useful for 3- and 4-color labeling with DyLight 405, Alexa Fluor® 488, and Alexa Fluor® 647 by using a confocal microscope equipped with a 405 nm laser and a krypton/argon laser. Fluorescence from RRX lies about midway between that of Alexa Fluor® 488 and Alexa Fluor® 647, and it shows little overlap with either dye. The krypton-argon laser emits lines at 488 nm, 568 nm, and 647 nm, which are optimal for exciting Alexa Fluor® 488, RRX, and Alexa Fluor® 647, respectively. By adding a 405 nm laser and a 420 nm emission filter, 4-color labeling is possible using DyLight 405-conjugated secondary antibodies from JIR. The separation between all four dyes is perfect for 4-color labeling, and all four dyes are very bright.
Rhodamine Red-X has good color separation from DyLight 405, and Alexa Fluor® 488, and Alexa Fluor® 647 for effective 4-color imaging.
Excitation and emission spectra of RRX-conjugated affinity-purified secondary antibodies, streptavidin, and purified proteins. Peak heights have been normalized, spectra were obtained with an M-Series spectrophotometer system from Photon Technology International, Inc. Values are approximate, actual values may vary depending on the spectrofluorometer used in each laboratory.
Emission spectra for optimal 4-color imaging using DyLight 405 (blue), Alexa Fluor® 488 (green), Rhodamine Red-X (red), and Alexa Fluor® 647 (purple).
|Excitation Peak||Emission Peak (nm)|
|Rhodamine Red-X, RRX||570||590|
Rhodamine Red™-X is a trademark of Molecular Probes, Inc.