By Inka, Ed. Brockhausen
A exotic panel of overseas specialists stories the significance of glycobiology, an interdisciplinary box encompassing chemistry, biochemistry, biology, body structure, and pathology. Glycobiology Protocols highlights very important methodological development that has been made within the box of glycobiology, assisting 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 curious about the biosynthesis of glycans. The tools defined for particular platforms might be converted for investigations of comparable biomolecules and tissues or mobilephone varieties. entire and hugely functional, Glycobiology Protocols presents researchers with an excellent foundation for learning the numerous elements of glycobiology with cutting-edge equipment. The protocols persist with the winning tools in Molecular Biology™ sequence structure, with each supplying step by step laboratory directions, an creation outlining the main at the back of the strategy, lists of kit and reagents, and pointers on troubleshooting and heading off identified pitfalls.
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Extra info for Glycobiology Protocols (Methods in Molecular Biology Vol 347)
0 with 1 M NaOH. The glycerol, detergent, and reducing agent should be added just prior to use. Dol-P-Man Synthase Assay 35 4. 1% (w/v) sodium dodecyl sulfate (SDS). The glycerol, detergent, and reducing agent should be added just prior to use. 5. 5- × 11-cm column, preferably plastic. 6. Sonifier (Branson model W185 or equivalent). 7. Amicon YM-10 ultrafiltration membranes (Millipore, Billerica, MA). 2. Enzyme Assay in Detergent Solution 1. , St. Louis, MO) in CHCl3ϺCH3OH (2Ϻ1). 2. GDP-[3H]mannose (50 µCi; 5–15 Ci/mmol, American Radiolabeled Chemicals,) and unlabeled 15 mM of GDP-Man (Sigma; see Note 5).
Biol. Chem. 278, 40,842–40,850. 30 Rush and Waechter 18. Orlean, P. , and Robbins, P. W. (1988) Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein. J. Biol. Chem. 263, 17,499–17,507. 19. , and Kinoshita, T. (1998) A homologue of Saccharomyces cerevisiae Dpm1p is not sufficient for synthesis of dolichol-phosphate-mannose in mammalian cells. J. Biol. Chem. 273, 9249–9254. 20. Colussi, P. , Taron, C. , Mack, J. , and Orlean, P. (1997) Human and Saccharomyces cerevisiae dolichol phosphate mannose synthases represent two classes of the enzyme, but both function in Schizosaccharomyces pombe.
1 mM Solution of the desired dolichyl monophosphate or polyisoprenyl monophosphate (for a description of the phosphorylation procedure, see Note 1) dispersed ultrasonically in 1% (w/v) CHAPS (see Note 2). 6. 0. 7. 1 M of MgCl2. 8. 1 M of 5′-adenosine monophosphate. 9. 9% (w/v) NaCl. 10. CHCl3/CH3OH (2/1, v/v). 11. 9% NaCl (3/48/47, v/v/v). 12. 1% Sodium dodecyl sulfate (SDS). 13. , Mount Prospect, IL). 14. Cellulose chromagrams sheets (Eastman Kodak, Rochester, NY). 15. , Washington, DC). 16. Packard TR2100 scintillation spectrometer.