DETECTION OF BIOTINYLATED PROTEINS IN CROSSED IMMUNOELECTROPHORESIS GELS - STUDIES ON PLATELET MEMBRANE-RECEPTORS AND MICROPARTICLES

Biotinylation can be used as an alternative for surface labeling of ce ll membrane proteins. The use of the water soluble N-hydroxysulfosucci nimide (NHSS)-biotin or the more lipophilic N-hydroxysuccinimide (NHS) -biotin reagent has been investigated in the present study labeling tw o central receptor complexes on the platelet surface, i.e. the glycopr otein (GP) Ib-IX and the GP IIb-IIIa complexes involved in platelet ad hesion and aggregation. Lack of labeling of the intracellularly locate d albumin was used as a negative control. The labeling has been studie d using crossed immunoelectrophoresis in the PhastSystem format after extraction of the labeled cells in Triton X-100, and it is shown that, using enzyme-conjugated avidin and chromogenic substrates, the biotin ylated proteins can be visualized directly in the dried electrophoresi s gel without the need for a transfer to a blotting membrane as is use d after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sui table conditions for biotinylation and for visualization in the crosse d immunoelectrophoresis gels are described. Further, surface-biotinyla tion of platelets was used to observe shedding of microparticles as a consequence of formation of the complement membrane attack complex. Fo r this purpose the formation and composition of the biotinylated micro particles were observed by flow cytometry and crossed immunoelectropho resis.