Glycobiology Protocols (Methods in Molecular Biology Vol by Inka, Ed. Brockhausen

By Inka, Ed. Brockhausen

A unusual panel of foreign specialists stories the significance of glycobiology, an interdisciplinary box encompassing chemistry, biochemistry, biology, body structure, and pathology. Glycobiology Protocols highlights very important methodological growth that has been made within the box of glycobiology, aiding scientists to respond to particular questions about glycoprotein buildings, at the biosynthesis of glycoconjugates, and at the services of lipid- or protein-bound carbohydrates. Glycobiology Protocols includes particular equipment for the research of the constructions or features of glycoconjugates, in addition to of glycosyltransferases and glycosidases fascinated about the biosynthesis of glycans. The equipment defined for particular platforms will be changed for investigations of comparable biomolecules and tissues or mobile forms. complete and hugely functional, Glycobiology Protocols presents researchers with a superb foundation for learning the various features of glycobiology with state of the art equipment. The protocols stick with the profitable tools in Molecular Biology™ sequence structure, with every one providing step by step laboratory directions, an creation outlining the main at the back of the approach, lists of kit and reagents, and tips about troubleshooting and keeping off identified pitfalls.

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3. 005 mL of enzyme and incubate at 25°C for 5 min in a water bath. 4. 005 mL of GDP-[3H]Man solution and mix on a vortex mixer, start stopwatch or timer, and immediately incubate at 25°C. 5. 45 mL of water-saturated butanol. 6. 6 mL of xylene-based scintillation fluid. Mix again on a vortex mixer for 10 s. 7. Centrifuge the vials for 2 min at 3000g in a tabletop centrifuge and then count in a scintillation counter (see Note 10). 8. Correct total activity by multiplying by 4/3 to account for sample volume counted in scintillation counter.

Phospholipids purchased from suppliers other than Avanti Polar lipids, as well as highly unsaturated phospholipids isolated from animal sources, are often not usable for activation of enzymes in the dolichol pathway because of their instability. Saturated phospholipids do not form bilayers or lipid matrices with the appropriate properties for the optimal activation of Dol-P-Man synthase. For initial studies, be sure to start with fresh solutions of plant PE. By comparison, solutions of unsaturated PC are relatively stable for several weeks, although it is recommended that all lipid solutions are stored under N2 at –20°C.

4968). Partial Purification of Man-P-Undec 15 Fig. 1. Proposed model for the synthesis and transbilayer movement of mannolipid intermediates involved in lipomannan assembly in Micrococcus luteus. Man-P-Undec and Man2-DAG are synthesized on the cytosolic monolayer of the bacterial plasma membrane from GDP-mannose and either Undec-P or diacylglycerol, respectively. The glycolipids are then translocated to the exoplasmic monolayer, where Man-P-Undec donates an average of 48 mannosyl units to Man2-DAG to form the full-length lipomannan.

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