methods and practical issues

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Ngày tải lên: 31/03/2014, 08:20

... 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-linked immunosorbent assays (ELISA). All these techniques have advantages and disadvan- tages. Solution methods ... serine proteases and esterases, including acetylcholinesterase, and should therefore be handled in a fume cupboard with extreme care! DFP is supplied in 1-g vials with a septum, and prior to dilution,...

Ngày tải lên: 08/10/2012, 10:25

... nature and limitations of the individual techniques, and (2) interpretation and reporting of mucin staining. 1.1. Nature and Limitations of Mucin-Staining Methods Mucin histochemistry, lectin, and ... mammary gland, defined by antibodies to cytokeratins. 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...

Ngày tải lên: 08/10/2012, 10:25

... Chemical, Lectin, and Antibody Methods Michael A. McGuckin and David J. Thornton 1. Introduction Detection 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 of secreted and membrane-associated mucins, respectively, and to Chapter 7 (4) for a discussion 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 as samples should be titrated for use as a standard curve, and samples should...

Ngày tải lên: 08/10/2012, 10:25

... poly- L -lysine for 5 min and dried at room temperature. 3. Methods 3.1. Observation and Measurement of Mucous Thickness on Unfixed Mucosal Sections ( see Notes 1–4) This is a rapid and simple method ... will be greatly reduced 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 feeding prostaglandin, N-acetyl-cysteine and other agents. Clin. Sci. 63, 187–195. 4. Bell, A. E., Allen, A., Morris, E. R., and Ross-Murphy,...

Ngày tải lên: 08/10/2012, 10:25

... samples no. 1 and 2 (values a and e): x = g/c × a/e (no dimension) Biosynthesis and Secretion of Mucin 65 65 From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: ... PhosphorImager. 4. The identification of mucin precursors and mature mucins using polypeptide-specific antisera and SDS-PAGE is elaborated in Chapters 20 and 21, and in several references (1,4–11). The precursor ... NJ 6 Quantitation of Biosynthesis and Secretion of Mucin Using Metabolic Labeling Jan Dekker, B. Jan-Willem Van Klinken, Hans A. Büller, and Alexandra W. C. Einerhand 1. Introduction Most epithelial...

Ngày tải lên: 08/10/2012, 10:25

... small gel, use 1.6 g of agarose and 160 mL of buffer, and for a large gel, use 2.8 g of agarose and 280 mL of buffer. 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 between band broadness and amount of sample to be loaded. Larger amounts of sample tend to yield poorer quality data; i.e., the bands become more ... other identified mucins. Separation and Identification of Mucin 77 7 Separation and Identification of Mucins and Their Glycoforms David J. Thornton, Nagma Khan, and John K. Sheehan 1. Introduction This...

Ngày tải lên: 08/10/2012, 10:25

... 2.400 a The values for ρ and η rel. are from ref. 14. Heterogeneity and Size Distribution of Gel-Forming Mucins 87 87 From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: ... variety of different sizes and should be chosen according to sample volume and concentration. Heterogeneity and Size Distribution of Gel-Forming Mucins 91 mucins on these grids; and the addition of ... K. E., and Chapman J. A. (1985) Mica sandwhich technique for preparing macromolecules for rotary shadowing. J. Ultrastruct. Res. 91, 66–76. 14. Kawahara K and Tanford C. (1966) Viscosity and density...

Ngày tải lên: 08/10/2012, 10:25

... 113 113 From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins Edited by: A. Corfield © Humana Press Inc., Totowa, NJ 10 Amino Acid Analysis of Mucins Jun X. Yan and ... 2 min for pump and column equilibration with the initial composition of the 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. References 1. Yan, J. X., Wilkins, M .R., Ou, K., Gooley, A. A., Williams, K. L., Sanchez, J C., Golaz, O., Pasquali, C., and...

Ngày tải lên: 08/10/2012, 10:25

... Kirk, M., Barnes, S., Lowary, T. L., and Hart, G. W. (1996) Selective detection and site-analysis of O-GlcNAc-modified glycopeptides by β-elimination and tandem electrospray mass spectrometry. ... Curmi, P. M. G., Williams, K. L., and Gooley, A. A. (1996) Sequencing Glycopeptides 121 121 From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins Edited ... 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)...

Ngày tải lên: 08/10/2012, 10:25

... 129 129 From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins Edited by: A. Corfield © Humana Press Inc., Totowa, NJ 12 Synthetic Peptides for the Analysis and Preparation of ... monoclonal antibodies (mAbs) and fragments have been produced 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 of significant features of an epitope that may contribute to antibody recognition and bind- ing (see Notes 10 and 11). 1.3. Purification...

Ngày tải lên: 08/10/2012, 10:25

... lysates and media. Nonspecific bands may be present and can be eliminated from consideration using the pSVL vector control lanes for comparison (Fig. 2A,B, lanes 1 and 3). The sizes of specific bands reported ... polymerase, and incubate the reaction at 72˚C to allow extension of the linked templates. Add 2 µg each of primers 5 and 6, and continue the PCR with 30 cycles at 94˚C for 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 two distinct domains....

Ngày tải lên: 08/10/2012, 10:25

... Nakamura, M., and Ohkura, Y. (1987) Fluoro- metric high performance liquid chromatography of N-acetyl- and N-glycolyl neuraminic acids 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 on From: 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 Capon Table 4 Elution Gradient for the Separation of Unreduced and Reduced Monosaccharides Time...

Ngày tải lên: 08/10/2012, 10:25

... 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 three column 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...

Ngày tải lên: 08/10/2012, 10:25

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

Ngày tải lên: 08/10/2012, 10:25

... 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 Packer 2. Materials 2.1. Solvents and Reagents 1. 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 method are similar for both methods. Although this method is not particularly sensitive by today’s analytical standards ( >1 nmol), it provides fast and reproducible...

Ngày tải lên: 08/10/2012, 10:25

Ngày tải lên: 08/10/2012, 10:25

Ngày tải lên: 08/10/2012, 10:25