Antibody Array Analysis with Label-based Detection and Resolution of Protein Size
Antibody Array Analysis with Label-based Detection and Resolution of Protein Size
复制标题
DOI：
10.1074/mcp.m800171-mcp200
复制
发表时间：
2009-02-01
影响因子：
7
通讯作者：
Lund-Johansen, Fridtjof
中科院分区：
文献类型：
--
作者：
Wu, Weiwei;Slastad, Heidi;Lund-Johansen, Fridtjof
DNA微阵列分析中的元素，如样品标记和捕获试剂的微斑点，已成功地用于可溶性细胞因子的多重测量。由于缺乏单一特异性抗体，以及许多蛋白质存在于复合体中，阻碍了在细胞生物学中的应用。在这里，我们结合了蛋白质印迹的原理，并确定了蛋白质大小作为额外的参数。来自不同细胞室的蛋白质被标记，并通过尺寸排除层析将其分离成20个组分。用复制的抗体阵列进行分析。所有抗体靶标的洗脱曲线被汇编成类似于Western blotts的彩色地图，带有大小分离范围(670-10 kDa)内的抗体反应性条带。为了处理大量的样品，开发了一种用于微孔板加工的新型固相。抗体被结合到蛋白G偶联微球上，表面标记了300种四种荧光染料的组合。用高速流式细胞仪检测颗粒色码和蛋白质标记的荧光。胞质蛋白激酶在可预测的洗脱点附近被检测到条带。对于具有非典型洗脱特征或多个上下文的蛋白质，使用两个或两个以上抗体作为特异性的内部参照。膜蛋白在空洞体积附近洗脱，并检测到与细胞内形式相对应的几个靶标的额外条带。细胞周期蛋白依赖性蛋白激酶(CDK)、细胞周期蛋白和细胞周期蛋白依赖性激酶抑制剂的洗脱曲线与它们在不同细胞周期阶段和亚细胞定位的复合体中的出现是一致的。二维平台避免了对单特异性捕获抗体的需要，并将抗体阵列分析的用途扩展到蛋白质复合体的研究。《分子与细胞蛋白质组学》8：245-257,2009。
Elements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines. Application in cell biology is hampered by the lack of mono-specific antibodies and the fact that many proteins occur in complexes. Here, we incorporated a principle from Western blotting and resolved protein size as an additional parameter. Proteins from different cellular compartments were labeled and separated by size exclusion chromatography into 20 fractions. All were analyzed with replicate antibody arrays. The elution profiles of all antibody targets were compiled to color maps that resemble Western blots with bands of antibody reactivity across the size separation range (670-10 kDa). A new solid phase designed for processing in microwell plates was developed to handle the large number of samples. Antibodies were bound to protein G-coupled microspheres surface-labeled with 300 combinations of four fluorescent dyes. Fluorescence from particle color codes and the protein label were measured by high-speed flow cytometry. Cytoplasmic protein kinases were detected as bands near predictable elution points. For proteins with atypical elution characteristics or multiple contexts, two or more antibodies were used as internal references of specificity. Membrane proteins eluted near the void volume, and additional bands corresponding to intracellular forms were detected for several targets. Elution profiles of cyclin-dependent kinases (cdks), cyclins, and cyclin-dependent kinase inhibitors, were compatible with their occurrence in complexes that vary with the cell cycle phase and subcellular localization. A two-dimensional platform circumvents the need for mono-specific capture antibodies and extends the utility of antibody array analysis to studies of protein complexes. Molecular & Cellular Proteomics 8:245-257, 2009.