By Michael A. McGuckin, David J. Thornton
Epithelial mucins are huge advanced mobile floor and secreted glycoproteins produced via mucosal epithelial cells. In, Mucins: equipment and Protocols specialist researchers within the box aspect the various equipment that are now familiar to review Mucins. those comprise tools and strategies for the simplest techniques to analysing every one particular zone of mucin biochemistry, body structure and biophysics prior to delivering person exact experimental protocols including troubleshooting and interpretation guidance. Written within the hugely winning tools in Molecular Biology™ sequence structure, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, with no trouble reproducible laboratory protocols, and key tips about troubleshooting and fending off identified pitfalls. Authoritative and functional, Mucins: equipment and Protocols is designed to be an invaluable source for these coming into the mucin box and to facilitate these already learning mucins to develop their experimental techniques to realizing mucosal biology.
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Additional resources for Mucins (Methods in Molecular Biology, v842)
Goodnow, C. , and McGuckin, M. A. (2008) Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis. PLoS Med 5, e54. 41. , Matthews, D. , Swallow, D. , Gum, J. , and Kim, Y. S. (1990) Assignment of the polymorphic intestinal mucin gene (MUC2) to chromosome 11p15. Ann Hum Genet 54, 277–285. 42. , Pratt, W. , Swallow, D. , Nguyen, V. , Aubert, J. , and Porchet, N. (1995) The MUC5AC gene: RFLP analysis with the Jer58 probe. Hum Genet 96, 367–368.
1 mM PMSF and allow to thaw if frozen. 2. Scrape the surface epithelium away from the underlying mucosa using a blunt instrument or a glass microscope slide. 3. Place the epithelial scrapings in at least five volumes of ice-cold 6 M guanidinium chloride extraction buffer and disperse with two or three strokes in a Dounce homogenizer with a loose pestle. 4. Cut the submucosal tissue (or whole tissue piece) into small pieces using scissors or a scalpel and submerge in liquid nitrogen. Pulverize or grind the tissue (we use a Retch tissue pulverizer, Retsch, Haan, Germany) for this purpose.
2 Gel-Forming and Cell-Associated Mucins… 33 2. Centrifuge secretions (4,400 × g, 4°C, 30 min). 3. Pour off and retain the supernatant for further purification. 2. Extraction of Gel-Forming Mucins from Tissue Samples Since tissue extracts contain high levels of cell debris and extracellular matrix fragments, as well as proteolyic enzymes, we always prepare mucins from this source under denaturing conditions. If mucins are to be prepared from the surface epithelium and submucosa separately, begin at step 1, whereas if they are to be extracted from the whole tissue piece, begin at step 4.
Mucins (Methods in Molecular Biology, v842) by Michael A. McGuckin, David J. Thornton