Glycosylation is one of the most common and important events in post-translational modification. Over half of all proteins are believed to be glycosylated, and the resulting glycoconjugates play an important role in many biological processes. They have been connected to instances of cancer development, retrovirus infection, and other diseases. In an effort to understand these diseases, glycoprotein analysis has become a growing area of research. (See examples of typical glycan structures.)
Analysis of glycoproteins requires sensitive and quantitative applications. LI-COR offers a single, optimized solution using the Odyssey® Systems and IRDye® labeled conjugates. This solution provides sensitive and quantitative results using two-color near-infrared detection at 700 nm and 800 nm wavelengths. Operating at this wavelength produces lower background from biological materials, buffer components, and standard membranes used in Western blotting and lectin binding applications and, thus, superior data.
Outlined below are a variety of applications for several one-color, visible glycoprotein applications that have been adapted to near-infrared fluorescence detection on an Odyssey Imaging System:
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Upgrade your current Odyssey or Pearl Imager software and all the benefits of this new, improved Image Studio Software.
If you would like to get an idea of how easy Image Studio Software is to use, try our free version – Image Studio Lite. Image Studio Lite allows you to analyze Western blot images but does not operate your LI-COR instrument to acquire images. Try it FREE – download now!
Biomolecule labeling continues to be a cornerstone feature of many in vitro and in vivo biological experiments. Click Chemistry has recently emerged as a convenient, versatile, and reliable method for labeling a wide variety of molecules for applications ranging from biomarker isolation to assay development.
LI-COR now offers a portfolio of Click Chemistry reagents for copper-catalyzed and copper-free methods. These products offer researchers flexibility to choose the correct reagent for a diverse array of applications. LI-COR Click Chemistry reagents include IRDye® 800CW, IRDye 680RD, and IRDye 650 near-infrared fluorescent dyes labeled with DBCO, azide, or alkyne groups.
Click Chemistry utilizes pairs of reagents that exclusively react with each other and are effectively inert to naturally-occurring functional groups such as amines. Unlike affinity interactions such as streptavidin-biotin, Click Chemistry forges covalent bonds between the reacting partners to deliver stable bioconjugates.
Click Chemistry reactions can be categorized into two separate groups, copper-catalyzed or copper-free. Copper-catalyzed Click Chemistry is used for initiating reactions between azides and alkynes. These reactions are also known as Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC). Although they initiate and accelerate Click Reactions, copper catalysts are cytotoxic and inappropriate for use in living systems.
For more sensitive detection – in the femtogram range – try WesternSure PREMIUM Chemiluminescent Substrate. The PREMIUM substrate is optimized for highly sensitive detection and long signal duration, making it ideal for use with imaging systems.
LI-COR now offers Click Chemistry reagents for copper-catalyzed and copper-free methods. One group of products within this portfolio includes IRDye® infrared dyes labeled with DBCO groups, which can be used for copper-free methods.
The dibenzocyclooctyne group (DBCO) allows copper-free Click Chemistry to be done with live cells, whole organisms, and non-living samples. DBCO groups will preferentially and spontaneously label molecules containing azide groups (—N3). Within physiological temperature and pH ranges, the DBCO group does not react with amines or hydroxyls, which are naturally present in many biomolecules. Reaction of the DBCO group with the azide group is significantly faster than with the sulfhydryl group (—SH, thiol).
BrightSite™ Optical Imaging Agents or probes developed using IRDye® infrared dyes can be used for in vitro, in vivo, and tissue imaging. This technology offers researchers the ability to take research from the cell to the animal, all within one lab.
Figure 1. Validation and Use of an IRDye Fluorescent Probe. After probe labeling, in vitro cellular assays and microscopy are used to confirm specificity. The desired target is then imaged in animals. Excised organs and tissues can be examined for more detailed localization of the probe. Animal image captured with Pearl Impulse. A more comprehensive discussion of approaches for the development of fluorescent contrast agents has also been published. Reference: Kovar, et al. Anal Biochem367(2007) 1-12.
Molecular imaging – achieved with near-infrared fluorescent technology from LI-COR!
WesternSure chemiluminescent reagents offer the best performance available when compared to other competitive products on the market. Both ULTRA and PREMIUM are highly sensitive enhanced substrates for detecting horseradish peroxidase (HRP) on immunoblots.
WesternSure HRP-conjugated secondary antibodies (Goat anti-Mouse and Goat anti-Rabbit) are compatible with a variety of chemiluminescent substrates and are optimized for use with WesternSure ULTRA and PREMIUM chemiluminescent substrates.
If you are in the US, check out the special March promotion for these New! Chemiluminescent Reagents (US and online orders only). Happy Blotting!
Are you doing chemiluminescent Western blots? Have you ever found yourself with a blot that is ready to image, but the darkroom is busy or the developer is broken?
FINALLY, you can image at your convenience. Keep your C-DiGit Blot Scanner on your lab bench, at your desk, or anywhere you choose (the image at the left shows just how small and portable the C-DiGit Scanner really is!).
The C-DiGit Chemiluminescent Western Blot Scanner maintains the simplicity of film exposures without the mess of the darkroom. You perform all of the same steps, without buying film and spending time in the darkroom. The C-DiGit Scanner gives you a complete digital replacement for film – keeping the advantages of film and eliminating many of the drawbacks – saving you time AND money!
Image Studio Lite is Western blot analysis software that is compatible with most image files and systems and is available as a FREE download from the LI-COR website.
Glycosylation is one of the most common and important events in post-translational modification. Over half of all proteins are believed to be glycosylated, and the resulting glycoconjugates play an important role in many biological processes. They have been connected to instances of cancer development, retrovirus infection, and other diseases. In an effort to understand these diseases, glycoprotein analysis has become a growing area of research. (See examples of typical glycan structures.)
