Detection of lectins using ligand blotting and polyacrylamide-type glycoconjugate probes

A sensitive and convenient method for detection of the carbohydrate-binding activity of lectins was established using the combination of blotting of lectins on polyvinylidene difluoride membranes, carbohydrate-conjugated biotinylated polyacrylamide-type probes (carbohydrate-bp probes), horseradish peroxidase-streptavidin, and detection by enhanced chemiluminescence of the enzyme reaction. This method was tested for detection of four plant lectins blotted on the membrane: concanavalin A was detectable down to 100 ng by mannose-bp probe, Ricinus communis agglutinin 120 to as low as 5 ng by N-acetyllactosamine-bp probe, soybean agglutinin to 1 microgram 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 detect recombinant human galectin-3 in Escherichia coli cell lysates and mannan-binding protein in human serum. These results indicate that this method is widely applicable to convenient detection and characterization of lectins in crude samples.