Glycoprotein Methods and Protocols: The Mucins by Julia R. Davies, Ingemar Carlstedt (auth.), Anthony P.
By Julia R. Davies, Ingemar Carlstedt (auth.), Anthony P. Corfield (eds.)
Anthony P. Corfield and a bunch of hands-on expermentalists collect in Glycoprotein tools and Protocols: The Mucins a cutting-edge selection of trustworthy and validated equipment for the examine of all elements of mucins. those strong equipment diversity from the coaching of mucins to their detection and quantitation with molecular organic and biochemical reagents. additionally incorporated are tools for the overview of mucin peptide and carbohydrate, of mucin biosynthesis and degradation, and of mucin bacteriology and cellphone biology. The thoughts reap the benefits of the most recent advancements in sensitivity and specificity of detection, and within the education of latest reagents for particular biochemical detection. Glycoprotein tools and Protocols: The Mucins bargains modern-day researchers a whole variety of simply reproducible analytical options for learning mucins. Its strong suggestions represent a launching pad for these getting into the sector for the 1st time and a state of the art assortment for these already lively in mucin research.
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Additional resources for Glycoprotein Methods and Protocols: The Mucins
Example text
Büller, H. , and Einerhand, A. W. C. (1998) MUC5B is the prominent mucin in human gallbladder and is also expressed in a subset of colonic goblet cells. Am. J. Physiol. 274, G871–G878. 18. , Axelsson, M. A. , and Hansson, G. C. (1995) The human MUC2 mucin apoprotein appears to dimerize before O-glycosylation and shares epitopes with the ‘insoluble’ mucin of rat small intestine. Biochem. J. 308, 873–880. 19. J. (1989) Isolation and structural analysis of rat gastric mucus glycoprotein suggests a homogenous protein backbone.
2. INCUBATION WITH ANTIBODY OR LECTIN 1. The concentration of antibody or lectin needs to be optimized to give the best signal-tobackground ratio. 1–10 µg/mL. 2. , 125I). 3. Lectins will need to be conjugated usually with biotin through either amino or carbohydrate groups; biotinylated lectins are commercially available. 4. It is recommended that replicate blots be probed with the same species/isotype irrelevant antibody to control for nonspecific binding. Incubation buffers need to contain protein (50% of blocking concentration) and/or nonionic detergent.
The sample/antibody mix can be preincubated (1–24 h at 4–37°C) prior to transfer to the mucin-coated plate. , step 7. 3. QUANTITATION 1. 7. 2. 1. 1. APPLICATION OF SAMPLES 1. 5 µL or added using a commercially available vacuum manifold device (these are preferable owing to more even sample distribution, 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 be included on all blots to determine interassay variation.



