Detection of lectin activity on western blots using erythrocytes.
A method is described for the direct detection of lectins, agglutinating erythrocytes, on nitrocellulose membranes after Western blotting, thus avoiding protein extraction from specific bands in the gel, followed by agglutination assays. The methodology essentially involves exposing the lectin band on a nitrocellulose strip to trypsinized rabbit erythrocytes (2%, in 0.15 M NaCl) for 30 min at 37 degrees C and then carefully transferring the membrane to saline (4 degrees C) for a few gentle 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 lectins that can agglutinate red blood cells and virtually has no cross reactivity with the various nonlectin proteins tested. Binding of erythrocytes to the lectin band on the nitrocellulose strip can be prevented by specific competing sugars. The method can be applied to screen for the presence of lectins in natural materials and to monitor lectin fractions during purification.
Rao
U J
UJ
Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore, India.
Ramasarma
P R
PR
Rao
D R
DR
Prasad
K V
KV
D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D003101:Collodion; D004912:Erythrocytes / Q000187:drug effects; D006386:Hemagglutination Tests; D037102:Lectins / Q000032:analysis*; D008358:Mannose; D008567:Membranes, Artificial; D011817:Rabbits; D012680:Sensitivity and Specificity; D014357:Trypsin
D037102:Lectins; D008567:Membranes, Artificial; D003101:Collodion; D014357:Trypsin; D008358:Mannose