STREPTAVIDIN BLOTTING - A SENSITIVE TECHNIQUE TO STUDY CELL-SURFACE PROTEINS - APPLICATION TO INVESTIGATE AUTOPHOSPHORYLATION AND ENDOCYTOSIS OF BIOTIN-LABELED INSULIN-RECEPTORS
STREPTAVIDIN BLOTTING - A SENSITIVE TECHNIQUE TO STUDY CELL-SURFACE PROTEINS - APPLICATION TO INVESTIGATE AUTOPHOSPHORYLATION AND ENDOCYTOSIS OF BIOTIN-LABELED INSULIN-RECEPTORS
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DOI：
10.1210/en.133.4.1803
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发表时间：
1993-10-01
期刊：
影响因子：
4.8
通讯作者：
TAYLOR, SI
中科院分区：
文献类型：
--
作者：
LEVYTOLEDANO, R;CARO, LHP;TAYLOR, SI
生物素的共价连接为标记细胞表面蛋白提供了一种有用的方法。经过生物素化后，用特定抗体进行免疫沉淀，然后用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法进行纯化。通过电印迹转移到膜上后，用标记的链霉亲和素探针可以检测到生物素标记的蛋白。该技术已被用于研究表达在小鼠NIH-3T3细胞表面的重组人胰岛素受体。胞外区的生物素化不会损害针对胞内区表位的抗体免疫沉淀胰岛素受体的能力。相反，生物素化降低了针对受体胞外区的多克隆抗体的亲和力。然而，通过增加抗受体抗体的浓度，成功地免疫沉淀生物素化受体是可能的。此外，生物素化受体保留了与胰岛素结合并经历胰岛素刺激的自磷酸化和内化的能力。使用酶标记的链霉亲和素可以使用化学发光技术来检测受体，从而消除了使用放射性的需要。就像这项技术对研究细胞表面胰岛素受体很有用一样，它也可以适用于研究其他细胞表面受体和蛋白质。
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.