Jackson ImmunoResearch Europe, Ltd

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

Tetramethyl Rhodamine Isothiocyanate
(TRITC), Rhodamine Red-X (RRX), and
Texas Red (TR)

Technical Service e-mail
tech@jireurope.com tech@jacksonimmuno.com

Conjugates of these rhodamine derivatives have different excitation (550, 570, and 596 nm) and emission (570, 590, and 620 nm) maxima (Table 1 and Figure 2). Although TRITC has been used traditionally with FITC for double labeling, better color separation is achieved by using RRX or Texas Red. However, it has been reported that use of Texas Red may lead to higher background staining (Wessendorf and Brelje, Histochemistry. 1992. 98, 81). We now recommend DyLight 594, instead of Texas Red, because it is brighter, more photostable, and more hydophilic than Texas Red.

Table 1. Approximate peak wavelengths of excitation and emission for JIR conventional and DyLight fluorophores conjugated to affinity-purified antibodies. Only approximate values are given for purposes of comparing one fluorophore with another. Actual values may vary depending on the spectrofluorometer used in each laboratory.

Fluorophore	Excitation Peak (nm)	Emission Peak (nm)
DyLight 405	400	421
Aminomethylcoumarin, AMCA	350	450
Cyanine, Cy2	492	510
DyLight 488	493	518
Fluorescein, FITC	492	520
DyLight 549	555	568
Indocarbocyanine, Cy3	550	570
Tetramethyl Rhodamine, TRITC	550	570
Rhodamine Red-X, RRX	570	590
DyLight 594	591	616
Texas Red, TR	596	620
Indodicarbocyanine, Cy5	650	670
DyLight 649	652	670

Excitation	Emission

Figure 1. Excitation and emission spectra of JIR fluorophore-conjugated, affinity-purified antibodies. This figure illustrates only the relative shape and position of each fluorophore in the peak region of its excitation and emission following conjugation to antibodies. Quantitative comparisons should not be made since peak heights have been normalized. For clarity, spectra for DyLight-antibody conjugates are not included here. For comparison, see DyLight-antibody spectra. All spectra were obtained with a M-Series spectrofluorometer system from Photon Technology International, Inc.

Rhodamine Red-X is particularly useful for three-color labeling with DyLight 488 and DyLight 649 by using a confocal microscope equipped with a krypton/argon laser. Fluorescence from RRX lies about midway between that of DyLight 488 and DyLight 649, and it shows little overlap with either dye (Figure 2). The krypton-argon laser emits lines at 488 nm, 568 nm, and 647 nm, all of which are optimal for exciting DyLight 488, RRX, and DyLight 649, respectively. By adding a 405 nm laser four-color labeling is possible using DyLight 405-conjugated secondary antibodies from JIR (Figure 2).

Figure 2. Emission spectra of DyLight 405 (blue), DyLight 488 (green), Rhodamine Red-X (red), and DyLight 649 (brown) conjugated to JIR affinity-purified secondary antibodies. This figure illustrates only the relative shape and position of each fluorophore in the peak region of its emission following conjugation to antibodies. Quantitative comparisons should not be made since peak heights have been normalized. All spectra were obtained with a M-Series spectrofluorometer system from Photon Technology International, Inc.