Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes
Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes
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DOI：
10.1186/s13039-017-0328-2
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发表时间：
2017-07-20
影响因子：
1.3
通讯作者：
Troadec, Marie-Berengere
中科院分区：
文献类型：
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作者：
Debaize, Lydie;Jakobczyk, Helene;Troadec, Marie-Berengere
背景：许多血液恶性肿瘤都存在遗传异常，包括染色体易位。从临床角度来看，染色体异常的检测不仅与诊断和治疗目的相关，而且与预后风险评估相关。从转化研究的角度来看，融合蛋白和蛋白质相互作用的鉴定为了解恶性肿瘤的发病机制带来了关键突破，从而在靶向治疗方面取得了重大成就。方法：我们描述了邻近连接试验（PLA）的优化，以确定融合蛋白的存在以及非贴壁前B细胞中的蛋白质相互作用。 PLA是一种创新的分子生物学蛋白质-蛋白质共定位检测方法，结合了显微镜的优点和分子生物学精度的优点，理论上能够检测0至40 nm范围内的蛋白质邻近度。结果：我们提出了一种优化的PLA程序。我们通过传统的细胞离心和优化缓冲液、改变孵育时间和修改洗涤步骤，克服了维持非贴壁血液细胞的问题。此外，我们提供令人信服的阴性和阳性对照，并证明优化的 PLA 程序对总蛋白水平敏感。优化的 PLA 程序可以检测非贴壁细胞上的融合蛋白和蛋白质相互作用。结论：此处描述的优化的 PLA 程序可以轻松应用于各种非贴壁血液细胞，从细胞系到患者细胞。优化的 PLA 方案能够检测融合蛋白及其亚细胞表达，以及非贴壁细胞中的蛋白质相互作用。因此，优化的PLA方案提供了一种可在生物领域广泛应用的新工具。
Background: Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy.Methods: We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm.Results: We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells.Conclusion: The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients' cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field.