By Linda Lindberg, Jining Liu, Jan Holgersson (auth.), Alain Beck (eds.)
Glyco-engineering is being constructed as a mode to regulate the composition of carbohydrates and to reinforce the pharmacological homes of monoclonal antibodies (mAbs) and different proteins. In Glycosylation Engineering of Biopharmaceuticals: equipment and Protocols, specialists within the box supply readers with construction and characterization protocols of glycoproteins and glyco-engineered biopharmaceuticals with a spotlight on mAbs. the amount is split in 4 complementary components facing glyco-engineering of healing proteins, glycoanalytics, glycoprotein complexes characterization, and PK/PD assays for healing antibodies. Written within the hugely winning 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 laboratory protocols, and tips about troubleshooting and heading off recognized pitfalls.
Authoritative and state-of-the-art, Glycosylation Engineering of Biopharmaceuticals: tools and Protocols serves as an excellent consultant for scientists striving to push ahead the fascinating box of engineered biopharmaceuticals.
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Additional resources for Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols
Store at −20°C in slidebox until use (see Note 3). 7. Count positive stained cells with regard to the total number of cells (based on nuclear staining). Some negative cells still remain at this stage even though cells are under selection. Estimating the number of stained cells allows the identification of wells suitable for further cloning. Save the wells that are best enriched in labeled cells. 4 Selection of Stable Cell Clones 1. Resistant cells are harvested and counted. Seed a single cell in each well of 96-well plates in the selection medium and incubate overnight at 37°C, 5% CO2.
16. 17. Nassimal El Maï et al. in healthy human serum. Xenotransplantation 9:376–381 Byres E, Paton AW, Paton JC, Lofling JC, Smith DF, Wilce MC et al (2008) Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin. Nature 456:648–652 Hedlund M, Padler-Karavani V, Varki NM, Varki A (2008) Evidence for a human-specific mechanism for diet and antibody-mediated inflammation in carcinoma progression. Proc Natl Acad Sci U S A 105:18936–18941 Lee EU, Roth J, Paulson JC (1989) Alteration of terminal glycosylation sequences on N-linked oligosaccharides of Chinese hamster ovary cells by expression of beta-galactoside alpha 2,6-sialyltransferase.
Trypsin/EDTA. 7. 2-, 5-, and 10 mL serological pipets. 3 Immunostaining 1. Fluorescence microscope: Filters should be selected to handle double or triple fluorochromes. 2. Fluorescent Sambucus Nigra agglutinin and prolong gold DAPI. The SNA lectin is specific for 6-linked sialic acid while DAPI is a specific labeling of the nucleus in cells. The procedure herein describes the use of SNA-FITC but any other fluorophore can be used. 3. DPBS (1×) with Ca2+ and Mg2+. 4. Cover glass and microslides. 5.