Distribution of cell surface saccharides on pancreatic cells. I. General method for preparation and purification of lectins and lectin-ferritin conjugates.
We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a "universal" affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.
D001665:Binding Sites; D002241:Carbohydrates / Q000032:analysis*; D002462:Cell Membrane / Q000032:analysis*; D055598:Chemical Phenomena; D002621:Chemistry; D005293:Ferritins; D037102:Lectins / Q000302:isolation & purification; D008722:Methods; D010179:Pancreas / Q000166:cytology
D002241:Carbohydrates; D037102:Lectins; D005293:Ferritins