O-GalNAc Modified Glycoprotein Assay Kit (FACS/Microscopy, Green Fluorescence) (ab235629)

Overview

  • Product name

    O-GalNAc Modified Glycoprotein Assay Kit (FACS/Microscopy, Green Fluorescence)
  • Sample type

    Adherent cells, Suspension cells
  • Product overview

    The O-GalNAc Modified Glycoprotein Assay Kit (FAC/Microscopy, Green Fluorescence)(ab235629) is a highly specific, simple and robust method for labeling and detection of O-GalNAc-glycosylated proteins within cells. The kit uses 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.

  • Notes

    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 1 structures 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.

  • Platform

    Flow cytometer, Fluorescence microscope

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 100 tests
    Copper Reagent (100X) 1 x 100µl
    Fixative Solution 1 x 10ml
    Fluorescent Alkyne (100X) 1 x 100µl
    GalAz Label (1000X) 1 x 10µl
    Permeabilization Buffer (10X) 1 x 25ml
    Reducing Agent (20X) 1 x 500µl
    Total DNA Stain (1000X) 1 x 20µl
    Wash Buffer (10X) 1 x 25ml

Images

  • Jurkat cells (1 X 10cells/ml) were cultured in presence of 1X GalAz Label for 24 hours at 37°C. Modified glycoproteins were detected according to the kit protocol and green fluorescence was analyzed by FACS (FL-1 channel). Negative control (white line), Background control (purple line), fluorescence corresponding to intracellular O-GalNAc-glycosylated proteins (green line).

  • Fluorescence Microscope images of cell surface (left panel) and subcellular localization (right panel) of O-GalNAc-glycosylated proteins in fixed HeLa cells.

Protocols

References

ab235629 has not yet been referenced specifically in any publications.

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