Janes Kevin A
Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA. E-mail:
Sci Signal. 2015 Apr 7;8(371):rs2. doi: 10.1126/scisignal.2005966.
Immunoblotting (also known as Western blotting) combined with digital image analysis can be a reliable method for analyzing the abundance of proteins and protein modifications, but not every immunoblot-analysis combination produces an accurate result. I illustrate how sample preparation, protocol implementation, detection scheme, and normalization approach profoundly affect the quantitative performance of immunoblotting. This study implemented diagnostic experiments that assess an immunoblot-analysis workflow for accuracy and precision. The results showed that ignoring such diagnostics can lead to pseudoquantitative immunoblot data that markedly overestimate or underestimate true differences in protein abundance.
免疫印迹法（也称为蛋白质印迹法）与数字图像分析相结合，可成为分析蛋白质丰度和蛋白质修饰的可靠方法，但并非每种免疫印迹分析组合都能产生准确结果。我阐述了样品制备、实验方案实施、检测方案和标准化方法如何深刻影响免疫印迹法的定量性能。本研究开展了诊断实验，以评估免疫印迹分析工作流程的准确性和精密度。结果表明，忽视此类诊断可能导致假定量免疫印迹数据，显著高估或低估蛋白质丰度的真实差异。
