Glycans are vital components of glycoproteins，glycolipids，and proteoglycans in all domains of life. Glycoproteins are grouped by the type of carbohydrate and amino acid linkage site. N-linked glycosylation is a modification of asparagine，whereas O-linked glycosylation occurs through the hydroxyl group of serine and threonine residues. Glycosylation occurs co- or post-translationally on >50% of eukaryotic proteins resulting in membrane-associated，intracellular，or secreted glycoproteins that are crucial in cellular processes，protein bioactivity and metabolic turnover. Modification by O-linked-N-acetyl glucosamine （O-GlcNAc） has rapidly emerged as a major cellular signaling mechanism with number of modified targets similar to protein phosphorylation. Many oncogenes and tumor suppressors are regulated by O-GlcNAcylation. O-GlcNAc modification is ubiquitous among eukaryotes，from yeast to humans and its modifying enzymes have been well characterized. O-GlcNAc modified nuclear and cytosolic targets include： transcription factors，signaling proteins，metabolic enzymes，mitochondrial trafficking，cell cycle regulation，glucose homeostasis. O-GlcNAc glycosylation is implicated in normal brain functions，etiology of neurodegeneration，type II diabetes，and pathways involved in morphogenesis and virulence factors of microbes and plant host cells. Since glycoproteins are not directly encoded in the genome，methods of characterization and analyses of glycoproteins are of great interest.Intended Use：This assay provides a convenient and accurate procedure to measure glycoproteins （O-GlcNAc Modified Proteins） in biological samples.Test Principle：The O-GlcNAc Modified Glycoprotein BioAssay Kit is a highly specific，simple and robust method for labeling and detection of O-GlcNAc-glycosylated proteins within cells. We use a modified glucosamine precursor that is fed directly into the cells，processed by the hexosamine pathway and incorporated into the proteins. Followed by click reaction with alkyne-containing dye，this system offers a powerful method for imaging the localization，trafficking，and dynamics of glycans，or detection by FACS for quantitative studies. Labeled Glycoproteins can be directly detected in 1D or 2D gels using the appropriate excitation sources，or enriched by immunoprecipitation with biotin-alkyne or antibodies prior to proteomic analysis. We provide sufficient materials for 100 assays in a 96-well plate format.Kit Components：Wash Buffer （10X），1x25mlFixative Solution，1x10mlPermeabilization Buffer （10X），1x25mlProtein Label （1000X），1x10ulCopper Reagent （100X），1x100ulFluorescent Azide （100X），1x100ulReducing Agent （20X），1x500ulTotal DNA Stain （1000X），1x20ul Storage and Stability：Store powder at 4°C liquid at -20°C. Store other components at 4°C. Stable for at least 6 months For maximum recovery of product，centrifuge the original vial after thawing and prior to removing the cap.
