Seeing Beyond the Visible With IRDye® Infrared Dyes
Harry L. Osterman and Amy Schutz-Geschwender
LI-COR Biosciences, 4647 Superior Street, Lincoln, NE 68504
Tel: 402-467-0730; Fax: 402-467-0819
E-mail: harry.osterman@licor.com
Introduction
Fluorescent dyes provide important labeling tools forbiological applications. Dyes emitting light at visible wavelengths are widely used for enzyme assays, pharmaceutical screening assays, and microscopy. The major scientific milestone of the last decade, the sequencing of the human genome, was made possible by fluorescent detection. The first automated system, commercialized by Applied Biosystems, was based on fluorescein- and rhodamine-related visible dyes1. A different approach was taken in the development of a near-infrared (NIR) fluorescence detection system using NIR dyes (now called IRDye® infrared dyes)2. This new detection system, first commercialized in 1993 by LI-COR® Biosciences, had inherent advantages resulting from the use of longer wavelength dyes, including wide dynamic range, reduced autofluorescent background, and very high sensitivity. The NIR sequencing system employing IRDye infrared dyes set new standards for read length and accuracy. As automated DNA sequencing continues to evolve with massively parallel methods and single molecule detection, fluorescence continues to play an important role in many of these systems. The CyDye® family of carbocyanine dyes developed in the late 1980's includes Cy3 (550/565 nm), Cy5 (649/670 nm), Cy5.5 (675/694 nm) and Cy7 (743/767 nm)3. The visible dyes Cy3 and Cy5 are widely used for DNA arrays, as well as many other DNA, protein, and microscopy applications.
The Alexa Fluor® family of visible dyes has also become popular in recent years. Although commonly used, visible fluorophores do not offer optimal performance for all applications. Cells, tissues, plastics, blotting membranes, and chemical compound libraries all possess intrinsic autofluorescence that can interfere with detection. However, in the near-infrared spectral region (650 - 900 nm), autofluorescent background is dramatically reduced. For this reason, NIR fluorophores, such as IRDye infrared dyes (Figure 1), are able to enhance detection sensitivity and signal-to-noise ratios in applications where autofluorescence had been limiting. This improvement has extended the benefits of fluorescent detection to new applications such as Western blotting and in vivo imaging, and can provide improved performance for cell-based assays, protein microarrays, microscopy, and screening of small molecule libraries.
Western Blotting
Historically, membrane applications such as Western blotting have been restricted to chemiluminescent and colorimetric detection because fluorescent methods have not provided sufficient sensitivity to detect endogenous protein levels. However, the lower autofluorescent background of nitrocellulose and PVDF membranes at NIR wavelengths allows fluorescence to be detected directly on membranes with sensitivity equivalent to or greater than chemiluminescent methods. A study comparing chemiluminescent detection to IRDye labeled secondary antibodies demonstrated that the NIR fluorescent method allowed quantification of proteins over a much broader linear range than chemiluminescence4. Use of direct fluorescence avoids the enzyme kinetics and substrate availability caveats inherent to chemiluminescent detection, producing more consistent results and a more quantitative measurement of protein levels. The ability to multiplex fluorophores in a two-color Western blot, thereby detecting and discriminating two protein targets simultaneously, is an added benefit that is not possible with chemiluminescence. A major application for two-color fluorescent Western blots is quantitative analysis of signal transduction pathways (Figure 2). Phosphorylation status of a protein of interest can be detected with phospho-specific antibodies using one fluorophore; a second spectrally distinct fluorophore is used to assess the total amount of that protein target, regardless of its phosphorylation state5, 6. Alternatively, the second color can be used to detect a different protein of interest or to normalize against a housekeeping protein.
Figure 2. Western blot analysis of ERK activation. ERK1/2 and phospho-ERK were detected simultaneously in lysates of unstimulated and EGF-stimulated A431 cells. Two-fold serial dilutions of lysate are shown. The single-color images can be overlaid to show both total ERK and phospho-ERK (yellow color indicates overlap of red and green signals). The mobility shift caused by phosphorylation can be seen in the EGF-stimulated lysate.
Protein Microarrays
Many types of protein microarrays can be detected with NIR fluorescence, and this can be particularly advantageous for arrays spotted on nitrocellulose surfaces. Nitrocellulose-coated glass slides are widely used for protein arrays due to their high protein binding capacity and ease of use, but the high autofluorescent background of these slides can limit their utility when combined with visible fluorophores. Background levels and signal-to-noise ratios have been compared for Cy3, Cy5, Alexa Fluor 680 and IRDye 800CW7. Because of the NIR advantage, Alexa Fluor 680 and IRDye 800CW showed the best sensitivity and broadest dynamic range, which can be important for detecting lower-abundance protein targets (Figure 3).
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