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.
Solum
N O
NO
Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, Norway.
Holme
P A
PA
Pedersen
T M
TM
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