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...

Ngày tải lên: 08/10/2012, 10:25

... buffer: 17.8 g of Na2HPO4·2H2O and 16.8 g of 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 ... and Hanisch, F-G. (1995)Studies on the order and site specificity of GalNAc transfer to MUC1 tandem repeats byUDP-GalNAc:polypeptide N-acetylgalctosaminyltransferase from milk or mammary car-ci...

Ngày tải lên: 08/10/2012, 10:25

... tetrazolium chloride and 1.65 mg of 5-bromo-4-chloro-3-indolyl-phosphate), and stop solution (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).Nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) ... sterilized.2. 20 mM benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside (benzyl-α-GalNAc) (Sigma),filter sterilized.3. Peptide-N 4-( acetyl-ß-glycosaminyl) asparagine amidase (N-gl...

Ngày tải lên: 08/10/2012, 10:25

... reversed-phaseHPLC Ultrasphere ODS (4.6 × 250 mm). 1, PA-Gal; 2, PA-Glc; 3, PA-Man; 4, PA-Rib; 5, PA-Fuc; 6, PA-Rham; 7, PA-ManNAc; 8, PA-deoxy-Rib; 9, PA-GlcNAz; 10, PA-GalNAc; 11, PA-NeuAc. Monosaccharide ... Michalski and CaponDifferent chromophores such as 2-aminopyridine (20), 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) (21), ethyl-4-aminobenzoate, and 4-aminobenzonitrile (22),h...

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... normal- or re-versed-phase high-performance liquid chromatography (HPLC) (8) or anion-exchange chro-matography, e.g., high-performance anion-exchange chromatography (HPAEC) on twoCarboPac PA-100 ... citrate-phosphate, pH 6.0; make up with HPLC-grade water.4. Dowex 50X-12 H+-form resin.5. Sephadex GM25.2.4. Hydrazinolysis1. 0.5-mL screw-capped V-bottomed Reacti-vials™ (Pierce and Warriner, C...

Ngày tải lên: 08/10/2012, 10:25

... protons; GalNAc-ol H-2, H-4, H-5 and H-6'Fig. 1 Analysis of Mucin-Type O-Linked Oligosaccharides 193atoms; Gal H-3 and H-4 atoms; Fuc H-5 and H-6 atoms; NeuAc H-3ax and H-3eqatoms; and CH3 of ... presence of -2 , 3- or -2 ,6-linked sialic acid is clearly shown bythe respective chemical shift of the H-3ax and H-3eq signals of the monosaccharides.The H-3ax and H-3eq re...

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... Supplies, Poole, Dorset, UK).5. 1 mM 2-sulfo-N-acetylglucosamine, 3-sulfo-N-acetylglucosamine, and 6-sulfo-N-acetylglucosamine, in water (see Note 3).2.2. Equipment1. High-performance liquid chromatography ... 2-, 3-, and 6-sulfo-N-acetylglucosamine in water may be aliquoted and stored at –20°C, and used as required.Fig. 1. Elution positions of acetate (CH3COO–), chloride (Cl–), s...

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... 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,Ale...

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... is important. Radioactive glucosamine is most com-monly used because it is incorporated into N-acetyl-D-glucosamine, N-acetyl-D-galac-tosamine, and the sialic acids, major monosaccharide components ... by addition of 1-mL of 10 mI Tris-HCl, pH 8.0, and load the prod-ucts onto columns of Sephadex G100, eluting with the same buffer and collecting 1-mLfractions. Metabolic Labeling Methods...

Ngày tải lên: 08/10/2012, 10:25

... (see Chapters 14–17), and (3) sulfate, which is incorporated in the trans-Golgistack and trans-Golgi network, as O-glycosylation elongation-terminator (see Chap-ters 14 and 17). Following the ... exposure timesto X-ray film to about one-tenth relative to autoradiography.18. Standard X-ray film (e.g., Fuji-RX) is not particularly sensitive. A more sensitive detec-tion of 35S,14C, and 3...

Ngày tải lên: 08/10/2012, 10:25