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 cell membrane proteins. The use of the water soluble N-hydroxysulfosuccinimide (NHSS)-biotin or the more lipophilic N-hydroxysuccinimide (NHS)-biotin reagent has been investigated in the present study labeling two central receptor complexes on the platelet surface, i.e. the glycoprotein (GP) Ib-IX and the GP IIb-IIIa complexes involved in platelet adhesion and aggregation. Lack of labeling of the intracellularly located albumin was used as a negative control. The labeling has been studied 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 biotinylated proteins can be visualized directly in the dried electrophoresis gel without the need for a transfer to a blotting membrane as is used after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Suitable conditions for biotinylation and for visualization in the crossed immunoelectrophoresis gels are described. Further, surface-biotinylation of platelets was used to observe shedding of microparticles as a consequence of formation of the complement membrane attack complex. For this purpose the formation and composition of the biotinylated microparticles were observed by flow cytometry and crossed immunoelectrophoresis.
Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, Norway.
N O
NO
Solum
P A
PA
Holme
T M
TM
Pedersen
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D001710:Biotin; D001792:Blood Platelets / Q000737:chemistry / Q000648:ultrastructure*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional / Q000379:methods* / Q000706:statistics & numerical data; D007202:Indicators and Reagents; D008565:Membrane Proteins / Q000097:blood*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000032:analysis; D019809:Streptavidin
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D007202:Indicators and Reagents; D008565:Membrane Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D001710:Biotin; D019809:Streptavidin; D016650:Fluorescein-5-isothiocyanate