IRDye® 680LT Outperforms
Alexa Fluor® 680
"Brightness" - Fluorescence intensity
IRDye 680LT matches or surpasses the brightness of Alexa Fluor® 680. Fluorescence determinations were made at a fixed antibody concentration of 10µg/mL in physiological buffer using dye-labeled goat anti-rabbit (GAR) conjugates prepared in-house. Fluorescence was measured using a PTI Fluorometer at the optimum excitation and emission wavelengths of each dye. The fluorescence intensity of each conjugate increased with increased degree of labeling. A plot of degree of labeling versus fluorescence (Shown Below) shows that the dynamic range for the 680LT GAR is much broader than the Alexa Fluor 680 which quickly levels off above D/P 3.4. The leveling off of the Alexa Fluor 680 conjugates is due to self-quenching. IRDye 680LT conjugates continue to increase in fluorescence intensity to at least D/P 6.4 which was the highest degree of labeling tested. Overall, IRDye 680LT is significantly brighter than Alexa Fluor 680.
The photostability of IRDye 680LT was compared to Alexa Fluor 680. Test samples were prepared by spotting equimolar amounts of each dye onto nitrocellulose membrane. The membrane was then scanned repeatedly on the Odyssey® Infrared Imaging System and the signal intensity was normalized to the control signal at time zero (Shown Below). The relative fluorescence of the IRDye 680LT exhibited greater photostability over Alexa Fluor 680.
Figure 1. Goat anti-rabbit IgG conjugates were analyzed at 10µg/mL in physiological buffer. Fluorescence was measured with a PTI fluorometer at optimum ex/em wavelengths for each dye.
Figure 2. Equimolar amounts of each dye (25 fmol) were spotted onto nitrocellulose membrane. The membrane was scanned repeatedly with the Odyssey® Classic Infrared Imaging System, and signal intensity was normalized to maximum intensity at time = 0.