Glycoprotein methods protocols - biotechnology 048-9-249-259 pdf
Glycoprotein methods protocols - biotechnology 048-9-001-013.pdf
... byAnthony P. CorfieldThe MucinsGlycoproteinMethodsand Protocols Isolation of Large Gel-Forming Mucins 33From :Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited ... Methods in Molecular BiologyTMHUMANA PRESSGlycoproteinMethodsand ProtocolsEdited byAnthony P. CorfieldVOLUME 125The MucinsMethods in Molecular BiologyTMHUMANA ... substi-tution, are resistant to proteolysis. Mucins comprise cell-associated, usually mono-meric species, as well as those that are secreted; the latter can be subdivided into large,gel-forming glycoproteins...
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Glycoprotein methods protocols - biotechnology 048-9-027-044.pdf
... 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 ... are, however, several other alternatives, such as 3-amino-9-ethylcarbazole (AEC), for use with horseradish peroxidase (HRP); 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT); ... G. (1984)Histochemical identification of 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,...
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Glycoprotein methods protocols - biotechnology 048-9-045-055.pdf
... 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, ... nonionicdetergents: 0.05% (v/v) Nonidet P-40 or Tween-20.4. Enzyme substrates: 2,2'-azino-bis(3-ethylbenzathiazaline 6-sulfonic acid) (ABTS) (1 mg/mL,A405nm), O-phenyldiamine (OPD) (1 mg/mL, A492nm), ... Histologically-based methods for detection of mucin,in Glycoprotein Methods and Protocols: The Mucins (Corfield, T., ed.), Humana,Totowa, NJ.2. Davies, R., Carlstedt, I. (2000) Isolation of large gel-forming...
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Glycoprotein methods protocols - biotechnology 048-9-057-064.pdf
... of phenol in 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 ... 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, 93–110.7. ... 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. B. (1984) Functional interac-tions of gastric mucus glycoprotein. ...
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Glycoprotein methods protocols - biotechnology 048-9-065-073.pdf
... 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 in ... 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 ... as [35S]sulfate-labeled, TCA-precipitable proteins (cpm/mL)(see Note 10).g. Mature [35S]sulfate-labeled, mucin band on 4% SDS-PAGE (au/mL).h. Total concentration of mucin by dot-blotting (au/mL).3.2.3....
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Glycoprotein methods protocols - biotechnology 048-9-075-085.pdf
... 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, ... 106) of the gel-formingmucins in particular, there are few separation techniques available to use with these77From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The ... assessment of pep-tide homogeneity is advisable. We analyze aliquots of the peaks by using matrix-assistedlaser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), andpurify...
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Glycoprotein methods protocols - biotechnology 048-9-087-096.pdf
... Heterogeneity and Size Distribution of Gel-Forming Mucins 8787From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana ... 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 ... Distributionof Gel-Forming MucinsJohn K. Sheehan and David J. Thornton1. IntroductionThe rheological properties of human mucus are dominated by the physical proper-ties of large secreted O-linked glycoproteins...
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Glycoprotein methods protocols - biotechnology 048-9-111-119.pdf
... Amino Acid Analysis of Mucins 113113From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., ... Wilkins, M. R., Yan, J. X., and Gooley, A. A. (1999) 2-DE spots amino acid analysis with9-fluorenylmethyl chloroformate, 2-D Protein Gel Electrophoresis Protocols (Link, A. J.,ed.), Humana, Totowa, NJ, ... mucin gly-coproteins. The hydrolysate of mucin is then derivatized with Fmoc on the α-aminogroups under alkaline conditions. The derivatized amino acids are separated by a C18reversed-phase column...
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Glycoprotein methods protocols - biotechnology 048-9-121-128.pdf
... 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 ... from Edman degrada-tion: the identification of Xaa-Pro-Xaa-Xaa as a motif for Thr-O-glycosylation. Biochem.Biophys. Res. Commun. 178, 1194–1201.6. Laursen, R. A. (1971) Solid-phase Edman degradation: ... arylamine-coated membranes are stable at –20°C; the N-ethyl-N'-dimethylamino-propylcarbodiimide (EDC reagent is stable at 4°C in a dry environment, and the buffer isstable at 4°C.2. Arylamine-derivatized...
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Glycoprotein methods protocols - biotechnology 048-9-129-141.pdf
... Synthesis on PinsSide chain protecting group Amino acidt-Butyl ether S, T, Yt-Butyl ester D, Et-Butoxycarbonyl K, H, W2,2,5,7,8-Pentamethylchroman-6-sulfonyl RTrityl CSynthetic Peptides for Antimucin ... citric acid monohydratemade up to 1 L with distilled water, pH 4.0.5. 2,2'-azino-bis[3-ethylbenz-thiazoline-6-sulfonic acid] (ABTS) substrate solution (Sigma):0.5 mg/mL in citrate phosphate ... contain-ing Tween-20 (0.1% v/v) for 10 min. Use fresh buffer for each wash.4. Dilute an appropriate horseradish peroxidase-labeled secondary antibody conjugate (e.g., horse-radish peroxidase-conjugated...
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