EZClick™ O-GalNAc Modified Glycoprotein Assay Kit (FACS/Microscopy, Green Fluorescence)

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. Attachment of O-linked mucin-type glycans is a common post-translational modification initiated by the addition of N-acetyl-galactosamine (GalNAc) to a Ser or Thr residue of newly assembled protein. Resulting GalNAc-(Ser/Thr) structure (Tn-antigen) can be further modified by the addition of either a sialic acid residue (sialyl-Tn), GlcNAc to form GlcNAc-GalNAc (core 3), or a galactose residue to form the GalGalNAc (core 1) structures respectively. The core 3 and 1structures are then further extended to form long and branched complex O-glycans. The core 1 (TF or T antigen) acts like oncofetal antigen, overexpressed in cancerous and precancerous conditions due to the Golgi apparatus disorder. GalNAc linked to the first mannose of glycosylphosphatidylinositol (GPI) core has been previously reported to be heterogeneously present on mammalian GPI-anchored proteins. Thus BioVision offers EZClick™ O-GalNAc Modified Glycoprotein Assay Kit, a highly specific, simple and robust method for labeling and detection of O-GalNAc-glycosylated proteins within cells. We use a modified galactosamine precursor that is fed directly into the cells, processed by the GalNAc salvage pathway to form the intermediate uridine diphospho (UDP)-GalNAz, which is recognized by GalNAc transferases in the Golgi and incorporated into the protein. 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.