Glycoprotein Methods and Protocols - P33

... solutions A and B at room temperature, and make solution C fresh daily.6. Make glycosidase assays using the following p-nitrophenyl substrates: N-acetyl α-D-galactosaminide, α-L-fucopyranoside, N-acetyl ... N-acetyl β-D-glucosaminide, and β-D-galacto-pyranoside, all prepared as 15 mM solutions in 0.01 M Tris buffer, pH 6.5. The stopsolution is a mixture of 0.5 M Na2CO 3and 0.5 M NaHCO3...

Ngày tải lên: 08/10/2012, 10:25

... 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...

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... 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-...

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... 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 Quantita...

Ngày tải lên: 08/10/2012, 10:25

... 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. Carbohydr...

Ngày tải lên: 08/10/2012, 10:25

... 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...

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... 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 exampl...

Ngày tải lên: 08/10/2012, 10:25

... 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...

Ngày tải lên: 08/10/2012, 10:25

... 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.,Gola...

Ngày tải lên: 08/10/2012, 10:25

... 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...

Ngày tải lên: 08/10/2012, 10:25