properties methods and collections

... a UV monitor and collect fractions using a fraction collector and subject to the appropriate carbohydrate and antibody analyses 3.6 Analysis of Mucins Methods for the detection and analysis of ... antibodies and lectins or coating methods such as the glycan detection method and enzyme-linked immunosorbent assays (ELISA) All these techniques have advantages and disadvantages Solution methods ... three principally different methods are available: solution assays such as colorimetric assays for hexose and sialic acid; membrane-based methods such as slot-blotting and staining with periodic...

Ngày tải lên: 08/10/2012, 10:25

... M and Milton, J D (1986) Lectin Methods and Protocols, Humana, Totowa, NJ Liener, I E., Sharon, N., and Goldstein, I J (1998) The Lectins: Properties, Functions, and Applications in Biology and ... mammary gland, defined by antibodies to cytokeratins J Cell Sci 75, 17–33 Kiernan, J A (1990) Histological and Histochemical Methods: Theory and Practice, Pergamon, Oxford, UK Andrew, S M and Jasani, ... such as Culling’s Handbook of Histopathological and Histochemical Techniques (including museum techniques) (36) or Kiernan’s Histological and Histochemical Methods: Theory and Practice (45) provide...

Ngày tải lên: 08/10/2012, 10:25

... core protein specificity), and lower sensitivity than slot-blot and immunoassay methods Chap- Detection and Quantitation of Mucins 47 ter (2) discusses these assays and they are mentioned here ... greater sample volume, and superior washing) 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 ... NJ Nagma, K., Thornton, D J., Khan, N., and Sheehan, J K (2000) Separation and identification of mucins and their glycoforms, in Glycoprotein Methods and Protocols: The Mucins (Corfield, T.,...

Ngày tải lên: 08/10/2012, 10:25

... and held together by bolts tightened by hand Millipore filter paper to act as a backing for the mucosa during sectioning and subsequent manipulation of the section 2.2 Histological Staining and ... (w/v) poly-L-lysine for and dried at room temperature Methods 3.1 Observation and Measurement of Mucous Thickness on Unfixed Mucosal Sections (see Notes 1–4) This is a rapid and simple method for ... layer will be greatly reduced and discontinuous Careful handling of the cryostat sections on the slide during fixing and staining is important because excessive washing and so on can remove mucus...

Ngày tải lên: 08/10/2012, 10:25

... PhosphorImager 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 ... Strous, G J and Dekker, J (1992) Mucin-type glycoproteins Crit Rev Biochem Mol Biol 27, 57–92 Van Klinken, B J W., Dekker, J., Büller, H A., and Einerhand, A W C (1995) Mucin gene structure and expression ... Einerhand, A W C., and Dekker, J (1995) Biosynthesis of intestinal mucins: MUC1, MUC2, MUC3, and more Biochem Soc Trans 23, 814–818 11 Van Klinken, B J W., Einerhand, A W C., Büller, H A., and Dekker,...

Ngày tải lên: 08/10/2012, 10:25

... a 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 Leave to cool before pouring (hand hot) and insert well-forming comb (see ... are 1.5 mm thick and 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 ... I., and Sheehan, J K (1996) Identification of glycoproteins on nitrocellulose membranes and gels Mol Biotechnol 5, 171–176 10 Thornton, D J., Howard, M., Devine, P L and Sheehan, J K (1995) Methods...

Ngày tải lên: 08/10/2012, 10:25

... Kadler, 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 ... (Fullerton, CA) SW 40Ti swing-out rotor, and finally unloading and analysis of the tube contents Measure out 18 mL of M GuHCl into one chamber of the gradient maker and an equal volume of M GuHCl into ... into 0.5-mL fractions, and these were analyzed for reactivity with antisera for the MUC5AC (᭹) and MUC5B (᭺) mucins The MUC5B mucin is more polydisperse than the MUC5AC mucin and has molecules of...

Ngày tải lên: 08/10/2012, 10:25

... 114 Yan and Packer chromatography, and leads to a significantly lower recovery of all the amino acids, especially serine and threonine Strong acid, high temperature, and short time acid ... at least for pump and column equilibration with the initial composition of the mobile phase Figures and show typical chromatograms of the separation of amino acid standards and hydrolyzed bovine ... samples and Dr Andrew Gooley and Prof Keith Williams for their support References Yan, J X., Wilkins, M R., Ou, K., Gooley, A A., Williams, K L., Sanchez, J.-C., Golaz, O., Pasquali, C., and Hochstrasser,...

Ngày tải lên: 08/10/2012, 10:25

... Notes and 8) and the length of sequence obtained is dependent on the number and distribution of proline residues Proline cleaves with reduced efficiency in Edman sequencing (Fig 1, vessel 1), and ... I., 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 Anal ... PerSeptive Biosystems and Beckman supply this product Quantitation of glycopeptides is quite difficult, and we recommend that 10% of the sample be first sequenced using the standard program prior...

Ngày tải lên: 08/10/2012, 10:25

... tandem repeat domain and overlapping each other by six amino acids (16), and the other used octamers overlapping each other by seven amino acids (17) Twelve antibodies were analyzed in total and ... information on antibody specificity and permits identification of significant features of an epitope that may contribute to antibody recognition and binding (see Notes 10 and 11) 1.3 Purification of ... must be pure and free from amines Several methods may be used to purify DMF (see Note 16) Piperidine 20% v/v: used for Fmoc deprotection Piperidine should be redistilled before use and made up...

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 and 3) The sizes of specific bands ... polymerase buffer, 2.5 mM MgCl2, 400 µM dNTPs, 200 ng of primers and or and 4, ng of template DNA (with only primers and 4), and U of Taq polymerase Perform a PCR program in a suitable thermal ... polymerase, and incubate the reaction at 72˚C to allow extension of the linked templates Add µg each of primers and 6, and continue the PCR with 30 cycles at 94˚C for 30 s, 50˚C for and 72˚C for...

Ngày tải lên: 08/10/2012, 10:25

... gas Program the oven temperature from 150 to 230°C and 3°C/min and then 230 to 250°C at 5°C/min Use injector and detector temperatures of 240 and 250°C, respectively Use an injection volume of ... Nakamura, M., and Ohkura, Y (1987) Fluorometric high performance liquid chromatography of N-acetyl- and N-glycolyl neuraminic acids and its application to their microdetermination in human and animal ... 164 Michalski and Capon Fig HPLC separation of sialic acid quinoxalinones obtained after mild acid hydrolysis of BSM Ac, acetyl; Lt, lactyl; Gc, glycolyl Methods 3.1 Release and Identification...

Ngày tải lên: 08/10/2012, 10:25

... (100 × cm) in a water jacket at 55°C and HPLC pump with refractive index and fluorescence detectors GlycoSepH™ and GlycoSepC™ HPLC column (Oxford GlycoSciences) Methods 3.1 Alkaline Borohydride Degradation ... 6.0, and load the sample carefully and directly to the center of the column, drop wise (see Note 3) Centrifuge for at 1100g and collect the excluded O-Glycanase O-Linked Chain Release and Fractionation ... (3 × 0.5 cm column) and elute with three column vol of HPLC-grade water Collect the effluent and eluent and then pool (2.5-mL volume) Load onto a PD10-Sephadex GM25 column and elute with HPLC-grade...

Ngày tải lên: 08/10/2012, 10:25

... 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 ... appearence of novel methods of ionization such as fast atom bombardment ionization (FAB), electrospray ionization (ESI), and matrixassisted laser desorption ionization (MALDI) Methods 2.1 Nuclear ... method was developed by Vliegenthart and colleagues during the 1970s and essentially applied to the structure determination of N-glycans of the Nacetyllactosamine and oligomannoside type (16) More...

Ngày tải lên: 08/10/2012, 10:25

... hygroscopic and must be thoroughly dried before weighing and making up as standards Standards of mM each of 2-, 3-, and 6-sulfo-Nacetylglucosamine in water may be aliquoted and stored at –20°C, and used ... chloride (Cl–), sulfate (HSO4–), and phosphate (H2PO3–) ions (see also Note 11) Chromatogram displays 10 nmol each of phosphate and sulfate, and trace amounts of chloride and acetate Note the difference ... 212 Harrison and Packer Materials 2.1 Solvents and Reagents Water, carbonate-free (see Note 1), and sparged with a constant flow of argon 0.1 M NaOH, made up with decarbonated water, and similarly...

Ngày tải lên: 08/10/2012, 10:25

... Subheading 2., item 5e at 37°C and 5% CO2 Caco-2 and A431 cells are passaged (trypsinised) and split 1:4 and the medium is changed every to d LS174T cells are split 1:2 and the medium changed daily ... only MUC and MUC2 at early passage, but at later passage and in later premalignant and malignant stages, they show de novo expression of MUC5AC, MUC5B, and MUC6 (Corfield, Myerscough, and Paraskeva, ... clumps of cells and replate under standard culture conditions Reattachment of cells may take several days, and during medium changing, any floating clumps of cells must be centrifuged and replated...

Ngày tải lên: 08/10/2012, 10:25

... nonradioactive medium Medium and washings are combined and mixed 1:1 (v/v) with M guanidine/PBS inhibitor cocktail (Subheading 2., items and 8) Metabolic Labeling Methods 231 Irrigate the flasks ... After incubation, remove the medium from the central well and wash the tissue and dish with mL of PBS Pool the medium and washings, and dialyze against three changes of L of M guanidine hydrochloride ... Electrophoresis and Vacuum Blotting (see Notes 16 and 17) Mix samples containing 10–500 mg mucin or >10,000cpm with 50 mL sample buffer and load onto horizontal 0.8–1% agarose gels Run Rainbow markers and...

Ngày tải lên: 08/10/2012, 10:25

... Einerhand, A W C., Büller, H A., and Dekker, J (1996) MUC2 is the prominent colonic mucin expressed in ulcerative colitis Gut 38, 554–563 Tytgat, K M A J., Bovelander, F J., Opdam, F J M., Einerhand, ... Einerhand, A W C., and Dekker, J (1995) Unpredictable behavior of mucins in SDS/polyacrylamidegel electrophoresis Biochem J 310, 1053,1054 23 Van Klinken, B J W., Einerhand, A W C., Büller, H A., and ... centrifugation the clear supernatant is collected, and the pellets are discarded (see Note 8) Take small aliquots (50–100 µL) of each homogenate and medium and add one-fourth volume of five-times concentrated...

Ngày tải lên: 08/10/2012, 10:25

... England Biolabs), containing 0.5 M sodium phosphate (pH 7.5) Nonidet-40 (New England Biolabs), 10% in water 10-times concentrated denaturing buffer (New England Biolabs), containing 5% SDS and ... are based on the methods to culture tissue and cell lines (see Chapter 18), methods for metabolic labeling of the mucin precursors (see Chapter 19), and methods to specifically immunoprecipitate ... is variation in the 256 10 11 12 13 14 15 16 Einerhand et al exact position of the precursor band on reducing SDS-PAGE, and sometimes double bands can be observed in particular individuals However,...

Ngày tải lên: 08/10/2012, 10:25

... E., and Louvard, D (1991) Use of cultured cells lines in studies of intestinal cell differentiation and function, in “Intestinal Absorption and Secretion.” Handbook of Physiology vol (Field, M and ... H A., and Einerhand, A W C (1995) Mucin gene structure and expression : protection vs adhesion Am J Physiol 269, (Gastrointest Liver Physiol 32), G613–G627 Lesuffleur, T., Zweibaum, A., and Real, ... control cells and in the threonine-free medium containing GalNAcα-O-benzyl of treated cells Then, the culture media are collected and centrifuged and the cells are lysed in mL of RIPA buffer and centrifuged...

Ngày tải lên: 08/10/2012, 10:25