The necessity of and strategies for improving confidence in the accuracy of western blots.
The necessity of and strategies for improving confidence in the accuracy of western blots.
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DOI：
10.1586/14789450.2014.939635
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发表时间：
2014-10
影响因子：
3.4
通讯作者：
Gomes AV
中科院分区：
文献类型：
--
作者：
Ghosh R;Gilda JE;Gomes AV
Western blotting是鉴定蛋白质和半定量蛋白质量最常用的实验室技术之一，然而，最近的一些研究结果表明，Western blotting可能不像以前假设的那样可靠。这并不奇怪，因为许多实验室没有意识到western blotting的局限性。在这篇手稿中，我们回顾了提高对western blots准确性的信心的基本策略。这些策略包括选择最佳归一化标准品，适当的样品制备，确定与感兴趣样品相关的抗体和蛋白质染色的线性范围，确认一抗的质量，防止信号饱和，准确定量目标蛋白的信号强度。虽然western blotting是一种强大的、不可缺少的科学技术，可以用来准确地定量相对蛋白水平，但有必要采用适当的实验技术和策略。
Western blotting is one of the most commonly used laboratory techniques for identifying proteins and semi-quantifying protein amounts, however, several recent findings suggest that western blots may not be as reliable as previously assumed. This is not surprising since many labs are unaware of the limitations of western blotting. In this manuscript we review essential strategies for improving confidence in the accuracy of western blots. These strategies include selecting the best normalization standard, proper sample preparation, determining the linear range for antibodies and protein stains relevant to the sample of interest, confirming the quality of the primary antibody, preventing signal saturation and accurately quantifying the signal intensity of the target protein. Although western blotting is a powerful and indispensable scientific technique that can be used to accurately quantify relative protein levels, it is necessary that proper experimental techniques and strategies are employed.