By Wenjie Peng, Corwin M. Nycholat, Nahid Razi (auth.), Inka Brockhausen (eds.)
Glycosyltransferases (GTs) are crucial for the biosynthesis of complicated glycoconjugates and are robust instruments to review the features of complicated glycans in overall healthiness, improvement and ailment. complicated glycoconjugates, similar to glycoproteins, proteoglycans and glycolipids, are assembled by means of GTs which synthesize particular linkages among sugars or sugars and protein. this is often unlike the non-specific or much less particular chemical glycation reactions, transglycosylation and opposite glycosylation reactions. Glycosyltransferases: equipment and Protocols encompasses a wide variety of reviews, equipment and protocols which shape a fantastic foundation for investigations of the function and mechanisms, biology and pathology related to GTs. Written within the winning Methods in Molecular Biology™ sequence layout, chapters comprise introductions to their respective issues, lists of the required fabrics and reagents, step by step, quite simply reproducible protocols, and notes on troubleshooting and averting recognized pitfalls.
Authoritative and simply obtainable, Glycosyltransferases: equipment and Protocols is an important contribution to glycobiology and glycopathology, and to functions of those enzymes in biotechnology and drug improvement. it is going to turn out valuable to scholars, postdoctoral fellows, and senior scientists wearing on study of GTs that has been intensified during the last years.
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Extra info for Glycosyltransferases: Methods and Protocols
Developed to determine enzymatic activity in the past few years ; however, many putative GTs remain uncharacterized since the number of predicated GTs is enormous. Few structural studies have been reported for this large family of enzymes. Only limited numbers of 3D structures from different GT families have been documented. Among these GTs, some catalyze the synthesis of secondary metabolites  and others mediate biogenesis of bacterial cell walls or polysaccharides [6, 7]. Several studies reported structures of bacterial GTs that are involved in protein glycosylation.
If necessary, you should bleach it based on the biohazard policy. 17. This final wash step includes four separate dishes with ddH2O, and the slides are dipped four times in each dish. 18. Avoid touching the print area. 19. The slide also can be dried under a gentle stream of ultra high purity nitrogen gas for 5 min. 20. Make sure the print surface is facing the laser source. 21. Each scan may lower the fluorescence of the signals. Glycosyltransferase Specificity Assay 13 22. Ensure that the enzyme amount matches with the slide label.
10. Analyze protein purity by running fraction samples on SDSPAGE (Fig. 2). Pool peak fractions and concentrate them to 30 mg/mL for crystallization screen subsequently (see Note 9). 3 Protein Enzymatic Activity Assay The substrates, metal ions (for metal-dependent GTs), and specific sugar donors are important elements for successful glycosyltransferase enzymatic assays. 1. The substrate of Gtf3 is GlcNAc modified Fap1 [10, 11]. Purify the substrate GlcNAc-modified Fap1 using glutathione sepharose beads from E.