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N-Glycan structure analysis using lectins and an alpha-mannosidase activity assay.
- Source :
-
Methods in enzymology [Methods Enzymol] 2006; Vol. 416, pp. 304-14. - Publication Year :
- 2006
-
Abstract
- Alpha-mannosidase IIx (MX) and alpha-mannosidase II (MII) are homologous enzymes whose critical roles in N-glycan processing were established in large part by analysis of the MII/MX double-knockout mouse. To analyze the structures of N-glycans synthesized in the mutant mice, we employed lectin blot and lectin histochemistry in addition to mass spectrometry analysis and two-dimensional high-performance liquid chromatography (HPLC) mapping. We also produced soluble MII and MX by transfecting mammalian cells with expression vectors and determined substrate specificity of MX. This chapter describes methods using lectins to analyze N-glycans in knockout mice and provides a protocol to assay alpha-mannosidase activity using soluble MX.
- Subjects :
- Animals
Animals, Newborn
COS Cells
Chlorocebus aethiops
Chromatography, High Pressure Liquid methods
Enzyme Activation
Mass Spectrometry
Mice
Mice, Knockout
Polysaccharides analysis
Substrate Specificity
alpha-Mannosidase genetics
alpha-Mannosidase isolation & purification
Histocytochemistry methods
Histocytological Preparation Techniques methods
Lectins chemistry
Polysaccharides chemistry
alpha-Mannosidase chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0076-6879
- Volume :
- 416
- Database :
- MEDLINE
- Journal :
- Methods in enzymology
- Publication Type :
- Academic Journal
- Accession number :
- 17113875
- Full Text :
- https://doi.org/10.1016/S0076-6879(06)16020-6