Eaton Samantha L, Hurtado Maica Llavero, Oldknow Karla J, Graham Laura C, Marchant Thomas W, Gillingwater Thomas H, Farquharson Colin, Wishart Thomas M
Division of Neurobiology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh.
Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh.
J Vis Exp. 2014 Nov 20(93):e52099. doi: 10.3791/52099.
The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term "western blot" suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many "optimized" western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.
20世纪70年代末，首次公开报道了蛋白质印迹法的使用，这是一种用于评估复杂生物样品中特定蛋白质的存在及相对丰度的技术。从那时起，蛋白质印迹法就成为分子生物学家实验方法中的常见组成部分。在PubMed上使用“蛋白质印迹法”一词进行粗略搜索表明，到2014年，超过22万篇已发表的手稿使用了该技术。重要的是，在过去十年中，技术成像取得了进展，同时开发出了灵敏的荧光标记，提高了灵敏度并产生了更大范围的线性检测。结果是现在有了一种真正基于可量化荧光的蛋白质印迹法（QFWB），使生物学家能够以前所未有的灵敏度和准确性进行比较表达分析。存在许多“优化”的蛋白质印迹法，并在不同实验室中使用。由于需要进行细微但未记录的程序修正，这些方法往往难以实施。本方案全面描述了一种成熟且可靠的QFWB方法，并配有故障排除策略。
