Taylor Sean C, Posch Anton
Bio-Rad Laboratories, 1329 Meyerside Drive, Mississauga, ON, Canada L5T 1C9.
Bio-Rad Laboratories GmbH, Heidemann Street 164, 80939 Munich, Germany.
Biomed Res Int. 2014;2014:361590. doi: 10.1155/2014/361590. Epub 2014 Mar 16.
Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.
蛋白质印迹法是一项已经应用了三十多年的技术，最初它是作为一种在复杂样品中检测蛋白质靶标的方法。尽管在成像和试剂技术方面已经取得了显著进展，以提高灵敏度、检测动态范围以及多重靶标检测的适用性，但基本技术本质上仍未改变。过去，蛋白质印迹法仅用于在复杂混合物中检测特定的靶标蛋白质，但现在期刊编辑和审稿人要求根据样品之间蛋白质表达的倍数变化对蛋白质印迹数据进行定量解释。这些计算基于印迹相关化学发光和/或荧光信号的差异光密度测定，而这现在需要在实验方法、数据采集和解释方面进行根本性的转变。我们最近发表了一种从蛋白质印迹中产生定量光密度数据的更新方法（泰勒等人，2013年），在此我们总结完整的蛋白质印迹工作流程，重点是定量蛋白质印迹的样品制备和数据分析。
