Glycoprotein Methods and Protocols - P16
Glycoprotein Methods and Protocols - P16
... lines LS174T, Caco-2 and A431, together produce all gastrointestinalmucins known at present: LS174T produces MUC1, -2 , -5 AC, -5 B, and -6 (12); Caco-2produces MUC1 and -3 (12) and A431 produces ... Biosynthetic Cell and Organ Culture Methods 21921918Biosynthesis of Mucin Cell and Organ Culture Methodsfor Biosynthetic StudyAnthony P. Corfield, Neil Myerscough,Alexandra W. C. Einerhand, B. Jan-Willem ... cell like), and Caco-2 (enterocyte like) (11,12).4. Collagen-coated flasks are necessary to obtain efficient attachment of primary culturesand some adenomas and carcinomas to the flasks, and to retain...
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Glycoprotein Methods and Protocols - P1
... byAnthony P. CorfieldThe MucinsGlycoproteinMethodsand Protocols Isolation of Large Gel-Forming Mucins 33From :Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited ... reactivity (᭜), absorbance at 280 nm (-- -- ) , and density ().12 Davies and Carlstedt7. The rotor type, speed and starting densities rather than the g-force are given for the runs.In our experience, ... antibodies andlectins or coating methods such as the glycan detection method and enzyme-linkedimmunosorbent assays (ELISA). All these techniques have advantages and disadvan-tages. Solution methods...
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Glycoprotein Methods and Protocols - P2
... T-antigen (β-d-Gal 1-3 GalNAc),but also to structures found within the backbone of oligosaccharides (β-d-Gal 1-3 /4GlcNAc) (19). Demonstration of PNA binding is not necessarily evidence of T-anti-gen ... mounting ,and its relative stability. There are, however, several other alternatives, such as 3-amino-9-ethylcarbazole (AEC), for use with horseradish peroxidase (HRP); 5-bromo-4-chloro-3-indolyl ... side chain substituted O-acetylated sialic acids: the PAT-KOH-Bh-PAS and the PAPT-KOH-Bh-PAS procedures. Histochem. J. 16, 623–639.8. Veh, R. W., Meesen, D., Kuntz, D., and May, B. (1982) Histochemical...
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Glycoprotein Methods and Protocols - P3
... presence of competitive inhibitors of O-glycosylation, such as benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside, without adversely affecting cells (9). With the eluci-dation of more sequence data for ... specificity (nonmucin glycoproteins can react, no carbohydrate or core pro-tein specificity), and lower sensitivity than slot-blot and immunoassay methods. Chap-Detection and Quantitation of Mucins ... on the blot (e.g., 3,3'-diaminobenzidine, 4-chloronapthol mix forHRP [14]; 5-bromo-4-chloro-3'-indolyphosphate toluidine salt, nitro blue tetrazolium chlo-ride mix for AP). Alternatively,...
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Glycoprotein Methods and Protocols - P4
... 70 mL of glycerol.8. Poly-L-lysine-coated slides: slides are immersed in 0.01% (w/v) poly-L-lysine for 5 minand dried at room temperature.3. Methods3 .1. Observation and Measurementof Mucous Thickness ... L. A., Allen, A., Morris, E., and Ross-Murphy S. B. (1988) Mucus glycoproteingels: role of glycoprotein polymeric structure and carbohydrate side-chains in gel-forma-tion. Carbohydrate Res. 178, ... rat, frog and human gastric mucosa: influence of feedingprostaglandin, N-acetyl-cysteine and other agents. Clin. Sci. 63, 187–195.4. Bell, A. E., Allen, A., Morris, E. R., and Ross-Murphy, S....
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Glycoprotein Methods and Protocols - P5
... homogenate and medium byWestern-type dot-blot procedure. Spot aliquots of the homogenates or media on nitrocel-lulose paper, using the dot-blot apparatus and air-dry for 5 min. Perform all ensuing pro-cedures ... [35S]sulfate-labeled, TCA-precipitable proteins in tis-sue homogenate (j1) and medium (j2) (cpm/mL) (Note 10).k. Mature [35S]sulfate-labeled mucin band in tissue homogenate (k1) and medium (k2)on 4% SDS-PAGE ... equiv-alent apparatus) to quantify the amount of radioactivity in an identified band on SDS-PAGE.6. Poly- or monoclonal antibodies (IgG-type) against nonglycosylated regions of the mucin-polypeptide...
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Glycoprotein Methods and Protocols - P6
... with“naked” cysteine-rich domains (4–7).Histological and biochemical investigations suggested that mucous-producing tis-sues and their secretions contained a complex mixture of mucin-type glycoproteins.However, ... prepared fromthe gel-forming mucins isolated from other epithelial secretions (respiratory and cer-vical) and from intestinal cell lines (HT-29 and PC/AA). In this example the electro-phoretic mobility ... monitored with a periodic acid-Schiff (PAS) assay and with avariety of lectins and mucin- or carbohydrate-specific antibodies (for a discussion ofthe relative merits and drawbacks of these analytical...
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Glycoprotein Methods and Protocols - P7
... Heterogeneity and Size Distribution of Gel-Forming Mucins 8787From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield ... MUC5B, and MUC6 (1–4)—are gel-forming mucins, and it is possiblethat there are others. In isolating a mixture of mucins from mucus, there is consider-able scope for heterogeneity in their mass and ... however, is rate-zonalcentrifugation, which separates molecules not on hydrodynamic volume alone but ontheir mass-to-volume ratio as well. The basis of rate-zonal centrifugation for this pur-pose is...
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Glycoprotein Methods and Protocols - P8
... 113113From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ10Amino Acid Analysis of MucinsJun X. Yan and Nicolle ... samplesand Dr. Andrew Gooley and Prof. Keith Williams for their support.References1. Yan, J. X., Wilkins, M .R., Ou, K., Gooley, A. A., Williams, K. L., Sanchez, J.-C.,Golaz, O., Pasquali, C., and ... HPLC system with a fluo-rescent detector and gradient controller is available.Acid hydrolysis is the first and most important step to release the amino acids fromthe proteins, and it must be carefully...
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Glycoprotein Methods and Protocols - P9
... window with elution profiles of PTH-threonine-GalNAc-Gal (black arrow-heads) and PTH-threonine-GalNAc (gray arrowheads) from the Applied Biosystems Procise. Stan-dard amino acids are shown for comparison ... Dixon, J. D., and Liang S.-P. (1991) Extending the perfor-mance of the solid-phase sequenator, in Methods in Protein Sequence Analysis (Jornvall,H., Hoog, J.-O., and Gustavsson, A.-M., eds.), Birkhauser ... has allowed mucin (andmucin-like) glycopeptides and trifluoromethane sulfonic acid (TFMSA)-treated mu-cin tandem repeats to be sequenced through the heavily glycosylated regions and themacroheterogeneity...
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