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Glycoprotein Methods and Protocols - P1

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... UV monitor and collect fractions using a fraction col-lector and subject to the appropriate carbohydrate and antibody analyses.3.6. Analysis of Mucins Methods for the detection and analysis ... antibodies and lectins or coating methods such as the glycan detection method and enzyme-linkedimmunosorbent assays (ELISA). All these techniques have advantages and disadvan-tages. Solution methods ... serine proteases and esterases, includingacetylcholinesterase, and should therefore be handled in a fume cupboard with extremecare! DFP is supplied in 1-g vials with a septum, and prior to dilution,...

Glycoprotein Methods and Protocols - P2

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... nature and limitations of the individual techniques, and (2) interpretation and reporting of mucinstaining.1.1. Nature and Limitations of Mucin-Staining Methods Mucin histochemistry, lectin, and ... mammary gland, defined by antibodies tocytokeratins. J. Cell Sci. 75, 17–33.45. Kiernan, J. A. (1990) Histological and Histochemical Methods: Theory and Practice,Pergamon, Oxford, UK.46. Andrew, ... Rhodes, J. M. and Milton, J. D. (1986) Lectin Methods and Protocols, Humana, Totowa,NJ.50. Liener, I. E., Sharon, N., and Goldstein, I. J. (1998) The Lectins: Properties, Functions, and Applications...

Glycoprotein Methods and Protocols - P3

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... Chemical, Lectin, and Antibody Methods Michael A. McGuckin and David J. Thornton1. IntroductionDetection and quantitation of mucins can be important in both the research and clinical settings. ... 1 and 2 (2,3) of this volume for the preparation ofsecreted and membrane-associated mucins, respectively, and to Chapter 7 (4) for adiscussion of methods for mucin separation.50 McGuckin and ... sample volume, and superior wash-ing). For quantitation and comparison across blots, a standard in the same buffer assamples should be titrated for use as a standard curve, and samples should...

Glycoprotein Methods and Protocols - P4

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... poly-L-lysine for 5 min and dried at room temperature.3. Methods 3.1. Observation and Measurementof Mucous Thickness on Unfixed Mucosal Sections (see Notes 1–4)This is a rapid and simple method ... will be greatlyreduced and discontinuous.7. Careful handling of the cryostat sections on the slide during fixing and staining is impor-tant because excessive washing and so on can remove mucus ... to 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,...

Glycoprotein Methods and Protocols - P5

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... samples no. 1 and 2 (values a and e):x = g/c × a/e (no dimension)Biosynthesis and Secretion of Mucin 6565From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: ... PhosphorImager.4. The identification of mucin precursors and mature mucins using polypeptide-specificantisera and SDS-PAGE is elaborated in Chapters 20 and 21, and in several references(1,4–11). The precursor ... NJ6Quantitation of Biosynthesis and Secretion of Mucin Using Metabolic LabelingJan Dekker, B. Jan-Willem Van Klinken,Hans A. Büller, and Alexandra W. C. Einerhand1. IntroductionMost epithelial...

Glycoprotein Methods and Protocols - P6

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... small gel, use 1.6 g ofagarose and 160 mL of buffer, and for a large gel, use 2.8 g of agarose and 280 mL ofbuffer.2. Leave to cool before pouring (hand hot) and insert well-forming comb (see ... 1.5 mm thick and 1 cm wide. There is a compromise betweenband broadness and amount of sample to be loaded. Larger amounts of sample tend toyield poorer quality data; i.e., the bands become more ... other identified mucins.Separation and Identification of Mucin 777Separation and Identificationof Mucins and Their GlycoformsDavid J. Thornton, Nagma Khan, and John K. Sheehan1. IntroductionThis...

Glycoprotein Methods and Protocols - P7

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... 2.400aThe values for ρ and ηrel. are from ref. 14.Heterogeneity and Size Distribution of Gel-Forming Mucins 8787From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: ... variety of different sizes and should be chosen according tosample volume and concentration.Heterogeneity and Size Distribution of Gel-Forming Mucins 91mucins on these grids; and the addition of ... K. E., and Chapman J. A. (1985) Mica sandwhichtechnique for preparing macromolecules for rotary shadowing. J. Ultrastruct. Res. 91,66–76.14. Kawahara K and Tanford C. (1966) Viscosity and density...

Glycoprotein Methods and Protocols - P8

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... 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 ... 2 min for pump and column equilibration with the initial composition ofthe mobile phase.5. Figures 1 and 2 show typical chromatograms of the separation of amino acid standards and hydrolyzed ... samples and 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...

Glycoprotein Methods and Protocols - P9

Danh mục: Môi trường

... Kirk, M., Barnes, S., Lowary, T. L., and Hart, G.W. (1996) Selective detection and site-analysis of O-GlcNAc-modified glycopeptides byβ-elimination and tandem electrospray mass spectrometry. ... Curmi, P. M. G., Williams, K. L., and Gooley, A. A. (1996)Sequencing Glycopeptides 121121From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited ... peptides.Acta. Chem. Scand. 4, 283–293.4. Edman, P and Begg, G., (1967) A protein sequencer. Eur. J. Biochem. 1, 80–91.5. Gooley, A. A., Classon, B. J., Marschalek, R., and Williams, K. L. (1991)...

Glycoprotein Methods and Protocols - P10

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... 129129From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ12Synthetic Peptides for the Analysis and Preparationof ... monoclonal antibodies (mAbs) and fragments have beenproduced by various methods. Some of these have found applications for radio-immunoscintigraphy and targeted therapy of cancer, and others have been ... information on antibody specificity and permits identification ofsignificant features of an epitope that may contribute to antibody recognition and bind-ing (see Notes 10 and 11).1.3. Purification...

Glycoprotein Methods and Protocols - P11

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... lysates and media. Nonspecificbands may be present and can be eliminated from consideration using the pSVL vectorcontrol lanes for comparison (Fig. 2A,B, lanes 1 and 3). The sizes of specific bandsreported ... polymerase, and incubate the reaction at 72˚C to allow extension of the linkedtemplates. Add 2 µg each of primers 5 and 6, and continue the PCR with 30 cycles at 94˚Cfor 30 s, 50˚C for 1 min and 72˚C ... 195–205.12. Bhargava, A. K., Woitach, J. T., Davidson, E. A., and Bhavanandan, V. P. (1990) Cloning and cDNA sequence of a bovine submaxillary gland mucin-like protein containing twodistinct domains....

Glycoprotein Methods and Protocols - P12

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... Nakamura, M., and Ohkura, Y. (1987) Fluoro-metric high performance liquid chromatography of N-acetyl- and N-glycolyl neuraminicacids and its application to their microdetermination in human and animal ... acid (Neu5Gc), and keto-deoxy-nonulosonic acid (Kdn), respectively. Substituents of the hydroxyl groups present onFrom: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: ... at 2°C/min.5. Use injector and detector temperatures of 240 and 250°C, respectively.174 Michalski and CaponTable 4Elution Gradient for the Separationof Unreduced and Reduced MonosaccharidesTime...

Glycoprotein Methods and Protocols - P13

Danh mục: Môi trường

... M. J. and Hounsell, E. F. (1998) HPLC and HPAEC of oligosaccharides and glyco-peptides, in Glycoscience Protocols (Hounsell, E. F., ed.), Humana, Totowa, NJ, pp. 79–100.14. Kakehi, K., and Honda, ... 0.5 cm column) and elute with threecolumn vol of HPLC-grade water.5. Collect the effluent and eluent and then pool (2.5-mL volume).6. Load onto a PD10-Sephadex GM25 column and elute with HPLC-grade ... cm)in a water jacket at 55°C and HPLC pump with refractive index and fluorescence detectors.5. GlycoSepH™ and GlycoSepC™ HPLC column (Oxford GlycoSciences).3. Methods 3.1. Alkaline Borohydride...

Glycoprotein Methods and Protocols - P14

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... or three atthe most) and the facilities that are available in the laboratory environment will defineFrom: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited ... carbons 4 and 5 and also the exocyclic chain ofsialic acid when it is not 8 or 9 substituted. The oligosaccharide-alditol is cleaved intwo fragments, one corresponding to the 3-branch and the ... was introduced in the 1970s and rapidlyreceived a large application for analyzing the sequence of N-acetyllactosamine- and oligomannosidic-type glycans.Originally, and before the development...

Glycoprotein Methods and Protocols - P15

Danh mục: Môi trường

... hygroscopic and must be thoroughly dried before weighing and making up as standards. Standards of 1 mM each of 2-, 3-, and 6-sulfo-N-acetylglucosamine in water may be aliquoted and stored at –20°C, and ... Harrison and Packer2. Materials2.1. Solvents and Reagents1. Water, carbonate-free (see Note 1), and sparged with a constant flow of argon.2. 0.1 M NaOH, made up with decarbonated water, and similarly ... equipment and the hydrolysis methodare similar for both methods. Although this method is not particularly sensitive bytoday’s analytical standards ( >1 nmol), it provides fast and reproducible...