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首页> 美国卫生研究院文献>BioMed Research International >Misleading Westerns: Common Quantification Mistakes in Western Blot Densitometry and Proposed Corrective Measures
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Misleading Westerns: Common Quantification Mistakes in Western Blot Densitometry and Proposed Corrective Measures

机译：误导性Westerns：Western印迹光度法中常见的定量错误和建议的纠正措施

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摘要

Densitometry data generated for Western blots are commonly used to compare protein abundance between samples. In the last decade, it has become apparent that assumptions underpinning these comparisons are often violated in studies reporting Western blot data in the literature. These violations can lead to erroneous interpretations of data and may contribute to poor reproducibility of research. We assessed the reliability of Western blot data obtained to study human myometrial tissue proteins. We ran dilution series of protein lysates to explore the linearity of densitometry data. Proteins analysed included αSMA, HSP27, ERK1/2, and GAPDH. While ideal densitometry data are directly proportional to protein abundance, our data confirm that densitometry data often deviate from this ideal, in which case they can fit nonproportional linear or hyperbolic mathematical models and can reach saturation. Nonlinear densitometry data were observed when Western blots were detected using infrared fluorescence or chemiluminescence, and under different SDS-PAGE conditions. We confirm that ghosting artefacts associated with overabundance of proteins of interest in Western blots can skew findings. We also confirm that when data to be normalised are not directly proportional to protein abundance, it is a mistake to use the normalisation technique of dividing densitometry data from the protein-of-interest with densitometry data from loading control protein(s), as this can cause the normalised data to be unusable for making comparisons. Using spiked proteins in a way that allowed us to control the total protein amount per lane, while only changing the amount of spiked proteins, we confirm that nonlinearity and saturation of densitometry data, and errors introduced from normalisation processes, can occur in routine assays that compare equal amounts of lysate. These findings apply to all Western blot studies, and we highlight quality control checks that should be performed to make Western blot data more quantitative.
机译：Western印迹产生的光密度测定数据通常用于比较样品之间的蛋白质丰度。在过去的十年中，很明显，在文献中报道Western blot数据的研究中经常违反支持这些比较的假设。这些违规行为可能导致错误的数据解释，并可能导致研究的可重复性差。我们评估了获得的用于研究人类子宫肌组织蛋白的蛋白质印迹数据的可靠性。我们进行了蛋白质裂解物的稀释系列试验，以探索光密度测定数据的线性。分析的蛋白质包括αSMA，HSP27，ERK1 / 2和GAPDH。尽管理想的光密度法数据与蛋白质丰度成正比，但我们的数据证实，光密度法数据经常偏离此理想值，在这种情况下，它们可以拟合非比例线性或双曲线数学模型，并且可以达到饱和。当使用红外荧光或化学发光在不同的SDS-PAGE条件下检测Western印迹时，观察到非线性光密度法数据。我们证实，与蛋白质印迹中目标蛋白质过量有关的重影伪影可能会歪曲发现。我们还确认，当要归一化的数据与蛋白质丰度不成正比时，使用归一化技术将目标蛋白的光密度法数据与加载对照蛋白的光密度法数据相除是错误的。可能会导致标准化数据无法用于比较。使用加标蛋白质的方式使我们能够控制每个泳道的总蛋白质量，而仅更改加标蛋白质的量，我们确认光密度法数据的非线性和饱和度以及归一化过程引入的误差会在常规分析中发生比较等量的裂解液。这些发现适用于所有Western blot研究，我们重点介绍应进行质量控制检查以使Western blot数据更定量。

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