Technical Center
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
Alexa Fluor® 488 493 519
Fluorescein, FITC/DTAF 492 520
Indocarbocyanine, Cy3 550 570
Tetramethyl Rhodamine, TRITC 550 570
Rhodamine Red-X, RRX 570 590
Alexa Fluor® 594 591 616
Texas Red, TR 596 620
Alexa Fluor® 647 651 667
Indodicarbocyanine, Cy5 650 670
Alexa Fluor® 680 684 702
Alexa Fluor® 790 792 803
 
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.

 

New Products
   

          Alexa Fluor® 680 and Alexa Fluor® 790
for Far-red and Infrared Detection on Western blots

 
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