Merck

Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology.

Bio-protocol (2017-07-20)
Ran Wang, Sumaira Z Hasnain
RESUMEN

Specialized secretory cells known as goblet cells in the intestine and respiratory epithelium are responsible for the secretion of mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 106 Da. These large proteins are densely substituted with short glycan chains, which have many important functional roles including determining the hydration and viscoelastic properties of the mucus gel that lines and protects the intestinal epithelium. In this protocol, we comprehensively describe the method for extraction of murine mucus and its analysis by agarose gel electrophoresis. Additionally we describe the use of High Iron Diamine-Alcian Blue, Periodic Acid Schiff's-Alcian Blue and immune-staining methods to identify and differentiate between the different states of glycosylation on these mucin glycoproteins, in particular with a focus on sulphation and sialylation.

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Sodium dodecyl sulfate, BioReagent, suitable for electrophoresis, for molecular biology, ≥98.5% (GC)
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Nitro Blue Tetrazolium, tablet
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Sodium chloride, BioXtra, ≥99.5% (AT)
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TWEEN® 20, viscous liquid
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N,N-Dimethyl-1,3-phenylenediamine dihydrochloride, 99%
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Iron(III) chloride, reagent grade, 97%
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Glycerol, for molecular biology, ≥99.0%
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Guanidine hydrochloride, for molecular biology, ≥99%
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Ethylenediaminetetraacetic acid, ACS reagent, 99.4-100.6%, powder
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Iodoacetamide, ≥99% (NMR), crystalline
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Schiff′s reagent
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Hematoxylin, certified by the Biological Stain Commission
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Bromophenol Blue