Isolation of integrin-based adhesion complexes.
Isolation of integrin-based adhesion complexes.
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DOI：
10.1002/0471143030.cb0908s66
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发表时间：
2015-03-02
影响因子：
--
通讯作者：
Humphries, Martin J
中科院分区：
文献类型：
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作者：
Jones, Matthew C;Humphries, Jonathan D;Byron, Adam;Millon-Fremillon, Angelique;Robertson, Joseph;Paul, Nikki R;Ng, Daniel H J;Askari, Janet A;Humphries, Martin J
细胞与其细胞外环境的整合通过细胞表面粘附受体（例如整合素）促进，其在正常发育和病理的发作中起重要作用。整合素与其在细胞外基质中的配体或其它细胞上的反受体的接合引发多种蛋白质在细胞内组装成粘附复合物，如焦点接触、焦点粘附和纤维状粘附。这些复合物中的蛋白质介导跨质膜的双向信号传导，因此有助于协调和/或调节细胞所经历的多种物理或化学信号。本单元中的方案描述了两种方法，用于分离或富集整合素相关粘附复合物中所含的蛋白质以及培养细胞中的局部质膜/胞质环境。在第一个方案中，使用包被有细胞外配体或抗体的微珠亲和分离整合素相关的粘附结构。第二个协议描述了腹侧膜制备物的分离，该腹侧膜制备物富含粘附复合物结构。该方案允许通过随后的下游分析通过蛋白质印迹法或质谱法测定粘附复合物组分。
The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signalling across the plasma membrane and as such help to coordinate and / or modulate the multitude of physical or chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes together with their local plasma membrane / cytosolic environments from cells in culture. In the first protocol integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components by subsequent downstream analysis by Western blotting or mass spectrometry.