Immobilization of pectin, xyloglucan and other soluble plant polysaccharides on blotting membranes.

Abstract

Soluble polysaccharides, of natural and synthetic origins, are immobilized by nylon, nitrocellulose, and polyvinylidene difluoride blotting membranes. Retention of an acid and a neutral polysaccharide, measured using radioactive pectin and xyloglucan-rich hemicellulose, exceeded 90% on some blotting membranes eluted in aqueous media. The two polysaccharides displayed different binding characteristics. Several polyanionic uronic acid polysaccharides, bound strongly to nylon 66 (Hybond N, Amersham) and to positively charge-modified or cationic nylon blotting membranes (Hybond N +, Amersham), but much less strongly to paper, nitrocellulose or polyvinylidene difluoride. Fragments of pectin, including Rhamnogalacturonan I and Rhamnogalacturonan II, and galacturonic acid oligosaccharides as small as the dimer also bind to nylon membranes, and can be detected using cationic dyes or by means of their radioactivity. Xyloglucan-rich hemicellulose in general binds more strongly than pectin to the same substrates. Of the substrates tested charge-modified nylon gave best retention of pectin, and paper gave best retention of xyloglucan during washes in water and solutions of salts, acids and bases. The influence of pH and solutions of mono, di- and trivalent salts on the retention of some pectic polysaccharides by nylon was investigated. A large proportion, in excess of 70% of pectin applied to charge-modified nylon, remained tightly bound at all salt concentrations up to 2 M. Soluble acid polysaccharides immobilized on blotting membranes could be detected by staining with cationic dyes, such as ruthenium red, alcian blue 8GX, and coriphosphine O, providing facile detection and a simple means of characterizing the cytochemical specificity of their staining reactions. Immobilized neutral polysaccharides, which do not react with cationic dyes, could usually be detected by the periodate-Schiff reaction, or, if labelled with radionuclides, by autoradiography or scintillation counting. Soluble polysaccharides, like proteins and nucleic acids, may therefore be immobilized on blotting membranes for investigation with cytochemical, immunocytochemical, and other molecular probing and detection procedures. Quantitative binding data showed marked differences in the affinity of different polysaccharides for blotting substrates. Detailed characterization of the binding behaviour is therefore a prerequisite for optimization and rational application of polysaccharide blotting.