Dot blot analysis of rat gastric mucin using histochemical staining methods.
Rat gastric mucins blotted on various membranes were detected using the periodate-Schiff staining method. The use of a polyvinylidene difluoride (PVDF) membrane gave the best results compared to nitrocellulose, nylon, positively charged nylon, and positively charged PVDF membranes. Alcian blue, high-iron diamine, galactose oxidase-cold thionin Schiff, and paradoxical concanavalin A stainings were also performed to detect mucins on PVDF membranes. Mucins were quantitatively detected by this analysis using each staining method with a range of detection from 0.02 to 1 microgram. Mucins extracted from rat gastric mucosa were detected by these dot blot assays at the position of void volume during Sepharose CL-4B chromatography. Mucins, after being purified with cesium trifluoroacetate centrifugation, were also mainly excluded from a Sepharose CL-2B column and detected by the dot blot assay using each staining method. In contrast, mucins after reduced alkylation were separated into two fractions during Sepharose CL-2B chromatography; one was excluded but the other was included in the column. Both fractions were detected using the periodate-Schiff method, but only the excluded fraction was stained using the paradoxical concanavalin A staining method. Thus, the dot blot assay using histochemical staining methods is useful for detecting individual mucins during gel chromatography.
Goso
Y
Y
Department of Biochemistry, Kitasato University School of Medicine, Sagamihara, Japan.
Hotta
K
K
D000818:Animals; D002847:Chromatography, Agarose; D005753:Gastric Mucosa / Q000737:chemistry*; D006651:Histocytochemistry / Q000379:methods*; D008297:Male; D008567:Membranes, Artificial; D009077:Mucins / Q000737:chemistry*; D017830:Octoxynol; D010503:Periodic Acid-Schiff Reaction; D011145:Polyvinyls; D051381:Rats; D017208:Rats, Wistar; D013194:Staining and Labeling / Q000379:methods; D005465:Fluorocarbon Polymers
D008567:Membranes, Artificial; D009077:Mucins; D017830:Octoxynol; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers