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 μg. 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.