Selective staining of human platelet glycoproteins using nitrocellulose transfer of electrophoresed proteins and peroxidase-conjugated lectins.
Platelet proteins (0.5-5 micrograms) were electrophoresed in a one-dimensional or an unreduced-reduced, two-dimensional sodium dodecyl sulfate gel system. The separated proteins were then transferred electrophoretically to nitrocellulose and reacted with peroxidase-conjugated lectins. Visualization of specific glycoproteins which bound the lectins was made by the chromogenic reaction catalyzed by peroxidase utilizing 3,3'-diaminobenzidine as the substrate. Wheat germ agglutinin specifically reacted with and allowed the visualization of glycoprotein Ib. Peanut agglutinin also specifically stained glycoprotein Ib after treatment of the nitrocellulose transferred proteins with neuraminidase. Ricinus communis agglutinin I stained thrombospondin, a 260 kDa protein, and factor VIII. Concanavalin A stained mainly glycoproteins IIb, III, IV, and V. Glycoproteins Ia, Ic, IIa, and other minor glycoproteins could be separated by unreduced-reduced, two-dimensional gel electrophoresis and were stained weakly with wheat germ agglutinin conjugates. These techniques were found to be reproducible as well as easily applied to the analysis and identification of platelet glycoproteins, particularly when dealing with a limited amount of platelets.
D000818:Animals; D001792:Blood Platelets / Q000032:analysis*; D003208:Concanavalin A / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000097:blood*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D037102:Lectins / Q000378:metabolism; D008722:Methods; D019887:Peanut Agglutinin; D037121:Plant Lectins; D011817:Rabbits; D013194:Staining and Labeling; D014909:Wheat Germ Agglutinins
D006023:Glycoproteins; D037102:Lectins; D019887:Peanut Agglutinin; D037121:Plant Lectins; C017707:Ricinus communis agglutinin-1; D014909:Wheat Germ Agglutinins; D003208:Concanavalin A; D006735:Horseradish Peroxidase