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使用未经验证抗体时蛋白质免疫印迹法的不准确性:需要蛋白质免疫印迹法最低报告标准(WBMRS)。

Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS).

作者信息

Gilda Jennifer E, Ghosh Rajeshwary, Cheah Jenice X, West Toni M, Bodine Sue C, Gomes Aldrin V

机构信息

Department of Neurobiology, Physiology, and Behavior, University of California Davis, Davis, CA, United States of America.

Department of Pharmacology, University of California Davis, Davis, CA, United States of America.

出版信息

PLoS One. 2015 Aug 19;10(8):e0135392. doi: 10.1371/journal.pone.0135392. eCollection 2015.

Abstract

Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.

摘要

蛋白质免疫印迹法是生物学研究中常用的技术。蛋白质免疫印迹法的一个主要问题不是该方法本身,而是使用了质量差的抗体以及使用了影响蛋白质免疫印迹线性和灵敏度的不同实验条件。对蛋白质免疫印迹中常用且经常修改的一些条件以及一些商业抗体进行调查发现,已发表的文章往往未报告重现结果所需的关键参数。这些参数包括上样的蛋白量、所用封闭液及条件、一抗和二抗的用量、抗体孵育液、检测方法以及所采用的定量方法。在本研究中,对大鼠心脏和肝脏样本中泛素化蛋白的比较显示,根据所使用的抗体不同结果也不同。使用纯化的泛素化蛋白、泛素链和游离泛素对五种商业泛素抗体进行验证表明,这些抗体在检测游离泛素或泛素化蛋白的能力上存在差异。使用六种市售抗体对年轻和老年大鼠心脏中经干扰素刺激基因15(ISG15)修饰的蛋白质进行研究表明,大多数抗体给出了不同的半定量结果,这表明抗体之间存在很大差异。本文提供了证据表明蛋白质免疫印迹缓冲液和所用抗体浓度的重要性。因此,迫切需要全面报告实验条件以提高蛋白质免疫印迹分析的准确性和可重复性。建议采用蛋白质免疫印迹最低报告标准(WBMRS)来提高蛋白质免疫印迹分析的可重复性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8d72/4545415/971cc47866dc/pone.0135392.g001.jpg

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