定量免疫印迹关键因素分析
An analysis of critical factors for quantitative immunoblotting.
作者信息
Janes Kevin A
机构信息
Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA. E-mail:
出版信息
Sci Signal. 2015 Apr 7;8(371):rs2. doi: 10.1126/scisignal.2005966.
Abstract
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.
摘要
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。
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