A Dual Array-Based Approach to Assess the Abundance and Posttranslational Modification State of Signaling Proteins
A Dual Array-Based Approach to Assess the Abundance and Posttranslational Modification State of Signaling Proteins
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DOI：
10.1126/scisignal.2002372
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发表时间：
2012-01-10
影响因子：
7.3
通讯作者：
Poetz, Oliver
中科院分区：
文献类型：
--
作者：
Luckert, Katrin;Gujral, Taranjit S.;Poetz, Oliver
对细胞信号的全系统分析需要检测和量化同一细胞样本中的许多不同的蛋白质及其翻译后修饰状态。在这里，我们介绍了两种基于阵列的小型化方法的协议，其中一种提供了关于中央信号蛋白的详细信息，另一种提供了周围信号网络的广泛特征。我们描述了基于微珠的阵列及其在表征β-连环蛋白的不同形式和功能方面的应用，以及裂解微阵列(反向蛋白质阵列)以及它们在检测和定量参与规范和非规范Wnt信号通路的蛋白质方面的应用。作为这种双重方法的应用，我们描述了细胞裂解物中β-连环蛋白信号的状态，并将这些分子特有的数据与信号通路的变化联系起来。这里描述的方案为细胞培养方法、珠子阵列和裂解微阵列提供了详细的说明，并概述了如何使用这些互补的方法在系统级别上洞察复杂的网络。
A system-wide analysis of cell signaling requires detecting and quantifying many different proteins and their posttranslational modification states in the same cellular sample. Here, we present Protocols for two miniaturized, array-based methods, one of which provides detailed information on a central signaling protein and the other of which provides a broad characterization of the surrounding signaling network. We describe a bead-based array and its use in characterizing the different forms and functions of beta-catenin, as well as lysate microarrays (reverse-phase protein arrays) and their use in detecting and quantifying proteins involved in the canonical and noncanonical Wnt signaling pathways. As an application of this dual approach, we characterized the state of beta-catenin signaling in cell lysates and linked these molecule-specific data with pathway-wide changes in signaling. The Protocols described here provide detailed instructions for cell culture methods, bead arrays, and lysate microarrays and outline how to use these complementary approaches to obtain insight into a complex network at a systems level.