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Affinity-based glycoprotein detection is an important part of the researches in glycosylation engineering. With advanced technologies and experienced scientists, Creative Biolabs has successfully developed a high-sensitive glycoprotein detection platform to offer the largest and most diverse portfolio of glycoprotein detection products or services. We can provide a series of affinity-based methods to determine whether a protein is glycosylated.
Glycoproteins have a wide range of physiological functions, including protein folding and trafficking, cell-cell and cell-matrix interaction, cellular differentiation, fertilization, immune response, the initiation and metastasis of tumors, etc. But, because of the low abundance of glycoproteins, the complexity of the glycan structures and the multiple substitutions (microheterogeneity) at glycosylation sites, glycoprotein analysis is very complicated and difficult.
We can provide a series of technologies for glycoprotein detection and analysis. Affinity-based glycoprotein detection methods have a wide spectrum of applications because they are more specific and they can facilitate the determination of the glycosylation type.
Fig.1 Workflow of immobilized-lectin affinity chromatography. (Hashim, 2017)
Lectins are carbohydrate-binding proteins that can specifically bind mono- or oligosaccharides to discriminate and analyze the glycan structures of glycoproteins. In the last decades, lectin-based applications for glycoprotein detection and characterization have been widely applied and it can detect glycoproteins separated by gel electrophoresis (SDS-PAGE) or two-dimensional gel electrophoresis (2D SDS-PAGE). During the lectin blotting procedure, protein samples are resolved on SDS-PAGE and transferred onto a nitrocellulose or polyvinylidene fluoride (PVDF) membrane, which is then incubated with a specific lectin and labeled with a group, such as digoxigenin (DIG) or biotin that will further bind to a secondary antibody or to avidin, subsequently, they are conjugated to an enzyme that catalyzes a color-producing reaction (alkaline-phosphatase) or a more sensitive luminescence-producing reaction (horseradish peroxidase).
Notably, when the lectin blotting method is combined with the high resolution and reproducibility of 2D SDS-PAGE and with the sensitivity of enhanced chemiluminescence, it can identify rapidly the glycoproteins of interest by comparison with a reference 2D SDS-PAGE protein map, and to obtain reliable and reproducible results.
Glycoproteins may be detected and isolated by specific enzymes, which has been employed for the improved detection of glycoproteins on blots. For instance, GALT (β-1,4-galactosyltransferase) can specifically add an azidogalactose to GlcNAc, that may be a marker for some proteins that are O-β-GlcNAcylated. O-GlcNAc can react with a fluorescent alkyne, leading to the detection by UV gel imager. Moreover, O-GlcNAcylated peptides can be labeled and further analyzed by mass spectrometry (MS).
The primary antibodies may specifically recognize some unique glycan moieties of glycoproteins, and they can react with secondary antibodies conjugated to enzymes that catalyze a color- or luminescence-producing reaction, which is exploited in glycoprotein detection with extraordinary high sensitivity and specificity.
Fig.2 Different approaches of enzyme-linked lectin assay (Hashim, 2017).
With years of experience, Creative Biolabs has successfully completed a lot of therapeutic glycoprotein detection projects. Because of the high sensitivity and specificity, affinity-based glycoprotein detection method helps us win a good reputation in our worldwide clients. Our scientists are committed to applying the high-quality products and services to meet your specific needs in glycoprotein project. The values of quality, timing and price is our basic criterion. If you are interested, please contact us without hesitation.