By Alexandra Castilho
Conceived with the purpose of delivering an array of suggestions and applied sciences presently in use for glyco-engineering designated dwelling organisms, this e-book features a wide variety of equipment being constructed to regulate the composition of carbohydrates and the houses of proteins via manipulations at the construction host instead of within the protein itself. the 1st 5 sections care for host-specific glyco-engineering and comprise chapters that supply protocols for changes of the glycosylation pathway in micro organism, yeast, insect, vegetation and mammalian cells, whereas the final sections discover replacement techniques to host glyco-engineering and chosen protocols for the research of the N-glycans and glyco-profiling through mass spectrometry. Written for the hugely profitable Methods in Molecular Biology sequence, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, effortlessly reproducible laboratory protocols and pointers on troubleshooting and heading off identified pitfalls.
Authoritative and large, Glyco-Engineering: tools and Protocols deals huge recommendations to assist researchers to decide on the expression method and method that most closely fits their meant protein study or applications.
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Additional resources for Glyco-Engineering: Methods and Protocols
5 Lectin Blotting Components 1. 0 g of Tris base in 800 mL of nanopure water, bring volume to 1 L and autoclave. 2. 05 % Tween-20 (TBST): Add 100 mL of 10× TBS to 900 mL of nanopure water. Add 500 μL Tween-20. 3. Albumin from bovine serum (BSA): 5 % (w/v) in TBST for blocking solution, 3 % (w/v) in TBST for lectin blotting. 4. 5 μg/ mL in 3 % BSA/TBST (or other lectin or antibody specific for the glycan of choice). 42 Anne A. Ollis et al. 5. Enhanced chemiluminescent Western blotting substrate (Pierce).
5. 100 % acetonitrile. 6. Reduction buffer: 10 mM Dithiothreitol (DTT) dissolved in 50 mM ammonium bicarbonate. 7. Alkylation buffer: 55 mM Iodoacetamide (IAA) dissolved in 50 mM ammonium bicarbonate. 8. Trypsin solution made using lyophilized trypsin protease, MS grade and 100 μL of 1 mM HCl and 900 μL of 40 mM ammonium bicarbonate in 9 % acetonitrile. 9. 5 % formic acid. 3 C-18 Cleanup 1. Pierce® C18 spin columns (Pierce) to remove contaminants from protein samples. 2. Activation solution: 50 % methanol, 50 % HPLC grade water.
Make up to 1 L with dH2O. 6. Terrific Broth: 12 g of tryptone, 24 g of yeast extract, 900 mL of dH2O. Mix until all components are in solution, then autoclave. 54 g of K2HPO4 to 90 mL of dH2O. Dissolve salts and make up to 100 mL with dH2O, then autoclave). 7. Qiagen plasmid midi kit (Qiagen). 8. 1 % (w/v) agarose gel. 9. Phusion® DNA Polymerase (New England Biolabs). P. Jaffé et al. 3 Glycan Production Screen and Genetic Mapping 1. Protran nitrocellulose membranes from Fisher Scientific. 1 Generation and Screening of Glyco Production Library Strain 4.