Sialic Acid (ManAz) Modified Glycoprotein Assay Kit (FACS/Microscopy, Green Fluorescence) (BN00683)

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BN00683
£530.10

Description

ELISA Kit Technical ManualMSDS

Sialic Acid (ManAz) Modified Glycoprotein Assay Kit (FACS/Microscopy, Green Fluorescence)

Glycans are vital components of glycoproteins, glycolipids, and proteoglycans in all domains of life. 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. Intracellular glycans mediate protein folding, stability, and trafficking while at the cell surface, they participate in recognition, cell-cell interactions involved in adhesion, migration, and embryonic development; host-pathogen interactions critical for bacterial and viral infections; and initiation of immune response. Aberrant glycosylation profiles correlate with inflammation and are universal feature of cancer, with sialic acids playing an especially prominent role as tumor associated carbohydrate antigens (TACAs). Altered sialylation of tumor cell surfaces is associated with several critical malignant properties that include invasiveness and metastatic potential suggesting its implication in clinical diagnosis. Since glycoproteins are not directly encoded in the genome, methods of characterization and analyses of glycoproteins are of great interest. Thus Assay Genie offers Sialic Acid (ManAz) Modified Glycoprotein Assay Kit, a highly specific, simple and robust method for labeling and detection of N-linked glycosylation of cell surface proteins. We use a modified mannosamine precursor that is fed directly into the cells, converted to sialic acid by the sialic acid biosynthetic machinery, and transported to the Golgi apparatus for glycan elaboration. 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 ManAz labeled glycans in proliferating cells. (A) Jurkat cells (1X10 6 cells/ml) were cultured in presence of 1X ManAz 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). Fluorescence in fixed and permeabilized cells (brown line) corresponds to surface and intracellular glycans whereas decreased signal in unprocessed cells resembles surface cell glycans (yellow line); negative control (black line). (B) Fluorescence Microscope analysis of cell surface glycans in non-fixed and non-permeabilized Jurkat cells (middle panel) and subcellular localization of ManAz modified glycans in fixed and permeabilized HeLa cells (middle panel). High resolution image (right panel) shows transported to the Golgi apparatus labeled glycans.

Key Information Description

Product SKU

BN00683

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 (Sialic Acid-Modified Proteins) in biological samples.

Detection Method

Flow Cytometry (Ex/Em 488/(530/590) nm) and Fluorescence Microscopy

Species Reactivity

Mammalian

Applications

This assay provides a convenient and accurate procedure to measure glycoproteins (Sialic Acid-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)
  • ManAz 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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