Lectin analysis of proteins blotted onto filters

Lectins are proteins that bind with great specificity to certain carbohydrate structures. Plant lectins are widely used for investigations of carbohydrate structure and for fractionation and purification of individual oligosaccharides and glycopeptides. This unit describes the use of lectins as sensitive indicators for the presence of certain carbohydrate structures linked to proteins blotted onto filters. A tagged lectin is incubated with a blot containing the target protein and binding of the lectin is detected by one of several different procedures. Direct approaches include using lectins labeled with 125I or conjugated to horseradish peroxidase or alkaline phosphatase, which can be detected by chromogenic or luminescent visualization systems. Indirect approaches involve using lectins conjugated to biotin or digoxigenin followed by a second incubation with alkaline phosphatase-conjugated avidin or antibodies specific for the haptenic digoxigenin group and then by visualization. Several commercial kits are available that provide labeled lectins, control proteins, and developing reagents needed for visualization. These systems can also be adapted for use with lectins other than those supplied with kits. The protocol in this unit is easy to perform with or without a kit. However, the results, while suggestive of carbohydrate structure, are not definitive.