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    • 23. 发明申请
    • A METHOD FOR RAPID FORMATION AND ISOLATION OF FOCAL ADHESION COMPLEXES
    • 一种快速形成和分离胶粘剂复合物的方法
    • WO1994025487A1
    • 1994-11-10
    • PCT/US1994004481
    • 1994-04-22
    • CHILDREN'S MEDICAL CENTER CORPORATION
    • CHILDREN'S MEDICAL CENTER CORPORATIONINGBER, Donald, E.PLOPPER, George, E.
    • C07K15/00
    • C07K14/7055C07K1/145C07K17/14
    • A method using microbeads coated with specific extracellular matrix ligands, for example, fibronectin or RGD-containing peptide, or any molecule that ligates integrins and thereby mediates attachment and spreading, even through receptors other than integrins, can be applied to the surface of microbeads to induce integrin clustering and isolate functional focal adhesion complexes (FACs) from eukaryotic cells such as mammalian, insect, or plant cells. In the preferred embodiment, magnetic microbeads are utilized. Bead-associated proteins are then isolated and collected for biochemical analysis using a combination of cytoskeletal extraction, sonication, dounce homogenization, and magnetic pelleting of the beads. The FACs are demonstrated to be functionally active and to contain a number of enzymatically and otherwise biologically active proteins. This method for FAC isolation facilitates analysis of molecular interactions and chemical and mechanical signaling mechanims within functionally intact FACs, independent of global changes in cells shape or cytoskeletal organization. It also serves as a means to isolate and identify proteins not previously known or known to be present in FACs, which can be used to form diagnostics such as peptides, nucleic acid probes or antibodies. The isolated FACs can also be used to screen for molecules which impact on the mechanical and chemical signalling systems within the FACs.
    • 使用包被特定细胞外基质配体的微珠,例如含纤连蛋白或含RGD的肽,或连接整联蛋白并由此介导附着和扩散的分子,即使通过非整联蛋白的受体也可以应用于微珠表面的方法 诱导整合素聚集并从真核细胞如哺乳动物,昆虫或植物细胞分离功能性粘附复合物(FAC)。 在优选实施例中,使用磁性微珠。 然后分离并收集珠相关蛋白用于生物化学分析,其使用细胞骨架提取,超声处理,均匀均匀化和珠粒的磁性沉淀的组合。 FAC被证明具有功能活性并且含有许多酶促和其它生物活性的蛋白质。 这种FAC分离方法有助于在功能完整的FAC中分子相互作用和化学和机械信号传导机制的分析,与细胞形状或细胞骨架组织的全局变化无关。 它还可用于分离和鉴定以前已知或已知存在于FAC中的蛋白质,可用于形成诊断例如肽,核酸探针或抗体。 分离的FAC也可用于筛选影响FAC内机械和化学信号系统的分子。