DETECTION OF LECTIN ACTIVITY ON WESTERN BLOTS USING ERYTHROCYTES

A method is described for the direct detection of lectins, agglutinati ng erythrocytes, on nitrocellulose membranes after Western blotting, t hus avoiding protein extraction from specific bands in the gel, follow ed by agglutination assays. The methodology essentially involves expos ing the lectin band on a nitrocellulose strip to trypsinized rabbit er ythrocytes (2 %, in 0.15 M NaCl) for 30 min at 37 degrees C and then c arefully transferring the membrane to saline (4 degrees C) for a few g entle washes and then fixing it in a solution (0.2% glutaraldehyde in 0.15 M NaCl) for 30 min. Later, the membrane is gently washed several times in 0.15 M NaCl containing 10 mM beta-alanine. The lectin band is visualized as a red agglutinated patch. The method is specific for le ctins that can agglutinate red blood cells and virtually has no cross reactivity with the various nonlectin proteins tested. Binding of eryt hrocytes to the lectin band on the nitrocellulose strip can be prevent ed by specific competing sugars. The method can be applied to screen f or the presence of lectins in natural materials and to monitor lectin fractions during purification.