Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS).
Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS).
复制标题
具有未验证抗体的蛋白质印迹不准确：需要蛋白质印迹最小的报告标准（WBMR）。
DOI：
10.1371/journal.pone.0135392
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Gomes AV
中科院分区：
文献类型：
--
作者：
Gilda JE;Ghosh R;Cheah JX;West TM;Bodine SC;Gomes AV
蛋白质印迹是生物学研究中常用的技术。Western印迹的主要问题不是方法本身，而是使用质量差的抗体以及使用影响Western印迹的线性和灵敏度的不同实验条件。对Western印迹中常用和经常修改的一些条件以及一些商业抗体的研究表明，已发表的文章通常未能报告重现结果所需的关键参数。这些参数包括上样蛋白的量、使用的封闭溶液和条件、使用的一抗和二抗的量、抗体孵育溶液、使用的检测方法和定量方法。在本研究中，大鼠心脏和肝脏样品中泛素化蛋白的比较显示出不同的结果，这取决于所使用的抗体。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，结果表明这些抗体检测游离泛素或泛素化蛋白的能力不同。调查蛋白质修饰干扰素刺激基因15（ISG15）在年轻和年老的大鼠心脏使用六个市售的抗体显示，大多数抗体给出不同的半定量结果，表明抗体之间的差异很大。证据显示的重要性的蛋白质印迹缓冲液和所使用的抗体的浓度。因此，迫切需要全面报告实验条件，以提高Western印迹分析的准确性和重现性。为提高免疫印迹分析的重复性，提出了一种免疫印迹最小报告标准（WBMRS）。
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.