O-GlcNAc Modified Glycoprotein Assay Kit (FACS/Microscopy, Green Fluorescence) (BN00934)

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BN00934
  • O-GlcNAc Modified Glycoprotein Assay Kit (FACS/Microscopy, Green Fluorescence) (BN00934)
  • O-GlcNAc Modified Glycoprotein Assay Kit (FACS/Microscopy, Green Fluorescence) (BN00934)
$662

Description

ELISA Kit Technical ManualMSDS

O-GlcNAc 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. 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. Thus Assay Genie offers O-GlcNAc Modified Glycoprotein Assay Kit, 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.

Figure: Analysis of metabolic labeling of GlcAz labeled glycans in proliferating cells. (A) Jurkat cells (1X10 6 cells/ml) were cultured in presence of 1X GlcAz 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-GlcNAc-glycosylated proteins (green line). (B) Fluorescence Microscope analysis of intracellular O-GlcNAc-glycosylated proteins in HeLa cells. High resolution images (middle and right panels) clearly show cytoplasmic and nuclear
localization of GlcAz modified glycans.

Key Information Description

Product SKU

BN00934

Size

100 assays

Kit Summary

  • Detection method- Flow Cytometry (Ex/Em 488/(530/590) nm) and Fluorescence Microscopy
  • Sample type- Adherent and suspension cells
  • #Species reactivity- Mammalian
  • Applications- This assay provides a convenient and accurate procedure to measure glycoproteins (O-GlcNAc Modified Proteins) in biological samples.

Detection Method

Flow Cytometry (Ex/Em 480/(530/590) nm) and Fluorescence Microscopy

Species Reactivity

Mammalian

Applications

This assay provides a convenient and accurate procedure to measure glycoproteins (O-GlcNAc Modified Proteins) in biological samples

Features and Benefits

Simple, fast, does not require lengthy incubation times

Kit Components

  • Wash Buffer (10X)
  • Fixative Solution
  • Permeabilization Buffer (10X)
  • GlcAz Label (1000X)
  • Copper Reagent (100X)
  • Fluorescent Alkyne (100X)
  • Reducing Agent (20X)
  • Total DNA Stain (1000X)

Storage Conditions

-20°C

Shipping Conditions

Gel Pack

USAGE

For Research Use Only! Not For Use in Humans.

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