Streptavidin blotting: a sensitive technique to study cell surface proteins; application to investigate autophosphorylation and endocytosis of biotin-labeled insulin receptors.
Covalent attachment of biotin provides a useful method to label cell surface proteins. Subsequent to biotinylation, the protein can be purified by immunoprecipitation with a specific antibody, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transfer to a membrane by electroblotting, the biotinylated protein can be detected by probing with labeled streptavidin. This technique has been used to investigate recombinant human insulin receptors expressed on the surface of murine NIH-3T3 cells. Biotinylation of the extracellular domain with an impermeant reagent did not impair the ability of an antibody directed against an epitope in the intracellular domain to immunoprecipitate insulin receptors. In contrast, biotinylation reduced the avidity of a polyclonal antibody directed against the extracellular domain of the receptor. Nevertheless, by increasing the concentration of the antireceptor antibody, it was possible to successfully immunoprecipitate the biotinylated receptor. Furthermore, biotinylated receptors retained the ability to bind insulin and undergo insulin-stimulated autophosphorylation and internalization. The use of enzyme-labeled streptavidin enables the use of chemiluminescence techniques to detect the receptors, thus obviating the need to employ radioactivity. Just as the technique is useful to study cell surface insulin receptors, it can be adapted to investigate other cell surface receptors and proteins.
Levy-Toledano
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R
Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Caro
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LH
Hindman
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N
Taylor
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SI
D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D001360:Avidin / Q000494:pharmacology; D001426:Bacterial Proteins; D001710:Biotin; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D007328:Insulin / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000187:drug effects / Q000378:metabolism*; D019809:Streptavidin; D014162:Transfection
D001426:Bacterial Proteins; D007328:Insulin; D008565:Membrane Proteins; D001360:Avidin; D001710:Biotin; D019809:Streptavidin; D011972:Receptor, Insulin