IRDye 680LT Infrared Fluorescent Dye is Bright and Photostable

IRDye® 680LT Infrared Dyes

Products

IRDye® 680LT NHS Ester

IRDye® 680LT Maleimide

Note: IRDye 680LT dye products should not be used for small animal in vivo imaging or In-Cell Western™ Assays. The higher level of fluorescent intensity creates high background making it unfavorable for use in these applications. We recommend using IRDye 680RD dye products for these applications.

IRDye 680LT dye is highly soluble in water and is significantly brighter and more photostable than many other 700 nm near-infrared dyes, such as Alexa Fluor® 680. The spectral characteristics of IRDye 680LT are well suited for use on LI-COR imaging systems with absorbance and emission maxima in aqueous solution and methanol of 676 and 693 nm, respectively. IRDye 680LT exhibits low background at near-infrared wavelengths resulting in higher signal-to-noise ratios.

IRDye 680LT dye conjugates can be used for quantitative Western blotting and fluorescence microscopy. When using IRDye 680LT secondary antibodies for Western blot detection, it is critical to follow the recommendations in the pack insert. Dilution optimization may be required to achieve best results.

IRDye Infrared Dyes Overview

Other Forms Available

IRDye 680LT NHS ester is available in labeling kits so that you can label your own compounds for assay or probe development.

IRDye 680LT dye-conjugated forms are available from LI-COR, including:

IRDye 680LT Dyes and Conjugates Used in Various Applications

Immunofluorescence staining of HER2 protein localization to SK-BR-3 cell membrane. Cells were cultured on coverslips. After fixation and permeabilization, cells were stained with rabbit anti-HER2 mAb (Cell Signaling Technology), IRDye 680LT Goat anti-Rabbit. Nuclei are stained with Sytox® Green dye. Images were acquired with an Olympus microscope and deconvolved with the accompanying software.
Example of multiplex Western blot detection. Lysates of EGF-treated A431 cells were separated and transferred to nitrocellulose. The blot was probed with anti-ERK and anti-phospho-ERK primary antibodies, and then detected with IRDye 680LT and IRDye 800CW secondary antibodies. Blot was imaged with Odyssey® Fc System for 2 min. This phospho-ERK antibody crossreacts with phospho-EGFR (upper green band).

IRDye 680LT Matches or Surpasses the Brightness and Photostability of Alexa Fluor 680

The fluorescence intensity of each conjugate increased with increased degree of labeling. Figure 1 shows that the dynamic range for IRDye 680LT Goat anti-Rabbit is much broader than that of the Alexa Fluor 680 dye-conjugate, which quickly levels off above D/P 3.4. Alexa Fluor 680 conjugates’ fluorescence intensities level off 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.

Figure 1. Comparison of fluorescence intensities of IRDye 680LT and Alexa Fluor 680. Fluorescence determinations were made at a fixed antibody concentration of 10 μg/mL in physiological buffer using dye-labeled goat anti-rabbit conjugates prepared in-house. Fluorescence was measured using a PTI Fluorometer at the optimum excitation and emission wavelengths of each dye.

The photostability of IRDye 680LT was compared to Alexa Fluor 680 in Figure 2. The relative fluorescence of the IRDye 680LT exhibited greater photostability over Alexa Fluor 680.

Figure 2. Photostability of IRDye 680LT and Alexa Fluor 680 Comparison. 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.