Lectin‐gradient gel affinity electrophoresis of glycoproteins

AbstractLectin‐gradient agarose gel affinity electrophoresis was developed for identification of glycoforms of glycoproteins in lectin affinity electrophoresis. Gradation of lectin was done by stacking agarose gel blocks with increasing concentrations of lectin (discontinuous system) and by keeping the plate in a moist chamber at 4°C overnight (continuous system) before electrophoresis. On the visualization of separated glycoform lines, the antibody‐affinity blotting was superior for low concentrations of α‐fetoprotein. Fluoresceine isothiocyanate (FITC) labeling of whole serum proteins, enzyme activity staining for alkaline phosphatase, and prestaining for lipoproteins were also applicable for visualization of proteins at higher concentrations. The conventional Western blotting can not be recommended because of the competition between lectin and glycoproteins in binding to nitrocellulose membrane. Lectin‐gradient affinity electrophoresis also had a wide application for optimization of the condition of lectin affinity electrophoresis.