DETECTION OF LECTINS USING LIGAND BLOTTING AND POLYACRYLAMIDE-TYPE GLYCOCONJUGATE PROBES

A sensitive and convenient method for detection of the carbohydrate-bi nding activity of lectins was established using the combination of blo tting of lectins on polyvinylidene difluoride membranes, carbohydrate- conjugated biotinylated polyacrylamide-type probes (carbohydrate-bp pr obes), horseradish peroxidase-streptavidin, and detection by enhanced chemiluminescence of the enzyme reaction. This method was tested for d etection of four plant lectins blotted on the membrane: concanavalin A was detectable down to 100 ng by mannose-bp probe, Ricinus communis a gglutinin 120 to as low as 5 ng by N-acetyllactosamine-bp probe, soybe an agglutinin to 1 mu g by beta-N-acetyl-D-galactosamine-bp probe, and wheat germ agglutinin to 5 ng by beta-N-acetyl-D-glucosamine-bp probe . All four lectins were detectable on an electroblotted membrane after SDS-polyacrylamide gel electrophoresis. This method was used to detec t recombinant human galectin-3 in Escherichia coli cell lysates and ma nnan-binding protein in human serum. These results indicate that this method is widely applicable to convenient detection and characterizati on of lectins in crude samples. (C) 1998 Academic Press.