Endo H Sensitivity
Endo H Sensitivity - Related Videos
07:46
Assessing Endothelial Vasodilator Function with the Endo-PAT 2000
0 Views •
Cited by 165 •
2010
A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.
02:58
Nanoparticle-Mediated Visualization of Endo-Lysosomal Remodeling in Bacterially-Infected Cells
0 Views •
2025
Source: Zhang, Y., et al. Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria. J. Vis. Exp. (2015)This video demonstrates the use of fluorescent nanoparticles to visualize endo-lysosomal remodeling in human epithelial cells infected by intracellular bacteria. Following bacterial invasion, the pathogens manipulate the host cytoskeleton and endo-lysosomal system, forming vacuoles with tubular projections that alter vesicle transport.
09:54
Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases
0 Views •
Cited by 31 •
2011
Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.
09:41
An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells
0 Views •
Cited by 29 •
2015
We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.
11:38
Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria
0 Views •
Cited by 3 •
2015
This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.
FAQs
Endo H sensitivity links glycan structure to secretory-pathway location. Glycoproteins that remain in the endoplasmic reticulum or early secretory pathway commonly retain cleavable high-mannose or hybrid N-glycans. Once processing in the Golgi produces mostly complex glycans, cleavage is generally lost, so the assay provides a biochemical indication of trafficking progress.
Cleavage depends on the oligosaccharide class attached to the glycoprotein. Endo H cuts within the chitobiose core of high-mannose and some hybrid N-glycans, whereas most complex N-glycans formed in the Golgi are not cleaved. Thus, the readout reflects glycan processing state rather than simply the presence of N-linked carbohydrate.
Protein synthesis alone does not establish secretory-pathway progression. A glycoprotein becomes largely Endo H resistant when its N-glycans undergo Golgi-associated processing into complex forms. Consequently, resistance supplies evidence that the protein advanced beyond an earlier compartment, while sensitivity remains consistent with retention in the endoplasmic reticulum or early secretory pathway.
A shift from sensitivity toward resistance indicates that the glycoprotein’s N-linked oligosaccharides changed from cleavable high-mannose or hybrid forms toward complex forms. This transition provides a biochemical marker of maturation through the secretory pathway. Following the change can therefore reveal whether processing proceeds normally or remains associated with an earlier intracellular stage.
A basic assay treats a protein sample with Endo H and then compares the treated material with the corresponding untreated material by electrophoresis. Cleavage alters the glycoprotein’s apparent electrophoretic mobility, allowing researchers to determine whether Endo H-sensitive oligosaccharides were present. This workflow converts glycan processing into a visible biochemical comparison.
Researchers use the readout to evaluate intracellular trafficking, determine whether a glycoprotein has progressed through the secretory pathway, or identify a trafficking defect. It also supports studies of protein maturation by monitoring whether glycan processing changes toward Endo H resistance, providing molecular evidence of maturation rather than relying only on protein detection.