Jackson ImmunoResearch

Technical Information on Probes Conjugated
to Our Antibodies and Other Proteins:

Rhodamine Red™ -X Conjugates

Technical Service e-mail
tech@jacksonimmuno.com

Rhodamine Red-X is a succinimidyl ester of Lissamine rhodamine B-labeled aminohexanoic acid. Rhodamine Red-X conjugates are superior to those of Lissamine rhodamine B in a number of respects. The reactive succinimidyl ester provides a more consistent conjugation and is less harmful to the activity and stability of proteins than the more highly reactive sulfonyl chloride on Lissamine rhodamine B reactive dye. Rhodamine Red-X conjugates also have a spacer arm (from aminohexanoic acid) which extends the dye out from the surface of the protein. Consequently, proteins conjugated with Rhodamine Red-X are significantly brighter than those conjugated with Lissamine rhodamine B.

Rhodamine Red-X conjugates are excited (570 nm) and emit light (590 nm) at the same wavelengths (see spectra below) as Lissamine rhodamine B, so no change of filter sets is required for those who currently use our Lissamine rhodamine conjugates. Rhodamine Red-X conjugates are particularly recommended for triple labeling using a confocal laser scanning microscope along with Cy2 and Cy5 because their fluorescence emission lies about midway between that of Cy2 and Cy5 and shows little overlap with these other fluorophores (see spectra below). Furthermore, the Krypton/Argon ion laser which is commonly used with confocal laser scanning microscopes emits lines of light at 488 nm, 568 nm, and 647 nm, all of which are optimal for excitation of Cy2, Rhodamine Red-X, and Cy5, respectively.


Excitation (EX) and emission (EM) spectra of Rhodamine Red-X conjugated antibody.	Emission spectra of cyanine- and Rhodamine Red-X (RRX)- conjugated antibodies.

Rhodamine Red-X may also be used for double labeling with Cy2 or for triple labeling with Cy2 and AMCA with an epi-fluorescent microscope using a mercury vapor lamp. (Cy 5 is not recommended for conventional epi-fluorescence microscopy because mercury vapor lamps do not emit any lines of light between 620 nm and 705 nm where Cy5 is optimally excited. Furthermore, the fluorescence emission from Cy5 is not very visible to the human eye.)

Rhodamine Red-X is available conjugated to all affinity-purified antibodies streptavidin, egg-white avidin anti-biotin anti-digoxin and ChromPure proteins. However, we will continue to sell Lissamine rhodamine B conjugates, if requested, but only until current inventory is sold out. After that, only Rhodamine Red-X conjugates will be available. The price and size of these products remains the same as those of Lissamine rhodamine B conjugates.

If you have any questions regarding Rhodamine Red-X, please notify us by e-mail.
Our staff scientists may be reached at tech@jacksonimmuno.com.