Glycosyltransferases: Methods and Protocols by Wenjie Peng, Corwin M. Nycholat, Nahid Razi (auth.), Inka

By Wenjie Peng, Corwin M. Nycholat, Nahid Razi (auth.), Inka Brockhausen (eds.)

Glycosyltransferases (GTs) are crucial for the biosynthesis of complicated glycoconjugates and are strong instruments to review the capabilities of advanced glycans in health and wellbeing, improvement and sickness. complicated glycoconjugates, reminiscent of glycoproteins, proteoglycans and glycolipids, are assembled through GTs which synthesize particular linkages among sugars or sugars and protein. this can be not like the non-specific or much less particular chemical glycation reactions, transglycosylation and opposite glycosylation reactions. Glycosyltransferases: tools and Protocols includes a wide selection of stories, equipment and protocols which shape a high-quality foundation for investigations of the function and mechanisms, biology and pathology related to GTs. Written within the profitable Methods in Molecular Biology™ sequence layout, chapters contain introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, conveniently reproducible protocols, and notes on troubleshooting and warding off recognized pitfalls.

Authoritative and simply obtainable, Glycosyltransferases: tools and Protocols is an important contribution to glycobiology and glycopathology, and to purposes of those enzymes in biotechnology and drug improvement. it's going to end up priceless to scholars, postdoctoral fellows, and senior scientists wearing on examine of GTs that has been intensified during the last years.

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Wash 20 μg substrate Fap1-GlcNAc bound to glutathione sepharose beads five times with the glycosylation buffer. 3. After the last wash, discard the supernatant and suspend the beads in 200 μL glycosylation buffer. 4. Add 2 μg of purified Gtf3 protein and 50 nM of UDP-[3H]glucose to the glycosylation reaction sequentially and incubate the mixture in an orbital shaker at 60 rpm for 1 h at 37 °C. Structural and Biochemical Analysis of a Bacterial Glycosyltransferase 35 5. Wash the beads three times with wash buffer and then transfer the beads to scintillation vials and mix with 5 mL scintillation cocktail.

Adv Enzymol Relat Areas Mol Biol 52:23–175 5. Pesnot T, Jorgensen R, Palcic MM, Wagner GK (2010) Structural and mechanistic basis for a new mode of glycosyltransferase inhibition. Nat Chem Biol 6:321–323 6. Sindhuwinata N, Munoz E, Munoz FJ, Palcic MM, Peters H, Peters T (2010) Binding of an acceptor substrate analog enhances the enzymatic activity of human blood group B galactosyltransferase. Glycobiology 20:718–723 7. Shih HW, Chen KT, Chen SK, Huang CY, Cheng TJ, Ma C, Wong CH, Cheng WC (2010) Combinatorial approach toward synthesis of small molecule libraries as bacterial transglycosylase inhibitors.

16. This incubation does not involve any reaction, but in such controlled studies it is important to conduct this incubation as for all other samples. 17. Repeat each assay with diluted samples: if the activity of T-synthase is very high, for example, the RFU is over 1,000,000 within 1 h reaction time, the sample needs to be diluted 1:10 and repeat the assay. This will provide more accurate information about the activity of T-synthase. 18. From the triplicate determinations, a standard deviation in activity of each sample can be obtained.

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