Christian Doppler Laboratory for Molecular Stress Research in Peritoneal Dialysis, Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, 1090 Vienna, Austria.
Division of Pediatric Nephrology and Gastroenterology, Department of Pediatrics and Adolescent Medicine, Comprehensive Center for Pediatrics, Medical University, 1090 Vienna, Austria.
J Proteome Res. 2020 Jun 5;19(6):2379-2390. doi: 10.1021/acs.jproteome.0c00061. Epub 2020 May 27.
Western blotting is widely used for protein identification and quantification in research applications, but different protein species, resulting from alternative splicing and post-translational modifications, can often only be detected individually by two-dimensional gel electrophoresis and immunodetection by Western blotting (2D-WB). The additional separation by isoelectric focusing enables the detection of different protein species with the same specific antibody. Reliable assignment of signals from antibody-based detection to the total protein spot pattern of the original gel image is a challenge in 2D-WB, often resulting in ambiguous results. We therefore propose a reliable strategy for assignment of antibody signals from 2D-WB to the total protein spot pattern, using an imaging workflow in combination with a straightforward and easily reproducible image alignment strategy. The strategy employs vector-based alignment of protein spots and image contours in a stepwise manner. Our workflow is compatible with various protein visualization techniques, including prelabeling of proteins and poststaining of gels and membranes, as well as with chemiluminescent and fluorescent detection of bound antibody. Here, we provide a detailed description of potential applications and benefits of our workflow. We use experimental test settings with gold-standard stressors in combination with multiple staining and detection methods, as well as spike-in recombinant proteins. Our results demonstrate reliable attribution of signals to very similar heat shock proteins, phosphorylation patterns, and global analysis of proteins modified with -linked -acetylglucosamine (-GlcNAc).
蛋白质印迹法广泛用于研究应用中的蛋白质鉴定和定量，但不同的蛋白质物种，由于选择性剪接和翻译后修饰，通常只能通过二维凝胶电泳和蛋白质印迹法（2D-WB）的免疫检测来单独检测。等电聚焦的额外分离使得能够用相同的特异性抗体检测不同的蛋白质物种。在 2D-WB 中，将基于抗体的检测信号可靠地分配给原始凝胶图像的总蛋白质斑点模式是一项挑战，通常会导致结果不明确。因此，我们提出了一种可靠的策略，用于将 2D-WB 中的抗体信号分配给总蛋白质斑点模式，该策略使用成像工作流程结合简单且易于重现的图像对齐策略。该策略采用基于向量的蛋白质斑点和图像轮廓的逐步对齐。我们的工作流程与各种蛋白质可视化技术兼容，包括蛋白质的预标记以及凝胶和膜的后染色，以及与结合抗体的化学发光和荧光检测。在这里，我们详细描述了我们的工作流程的潜在应用和优势。我们使用具有金标准应激物的实验测试设置，结合多种染色和检测方法以及掺入的重组蛋白。我们的结果证明了信号与非常相似的热休克蛋白、磷酸化模式以及用连接的 N-乙酰葡萄糖胺（-GlcNAc）修饰的蛋白质的全局分析的可靠归属。
