Abstract:
: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.
journal_name
Biomed Res Intjournal_title
BioMed research internationalauthors
Butler TAJ,Paul JW,Chan EC,Smith R,Tolosa JMdoi
10.1155/2019/5214821subject
Has Abstractpub_date
2019-01-21 00:00:00pages
5214821eissn
2314-6133issn
2314-6141journal_volume
2019pub_type
杂志文章abstract::Our previous studies have demonstrated that diabetes-induced oxidative stress alters homeostasis of retinal nerve growth factor (NGF) resulting in accumulation of its precursor, proNGF, at the expense of NGF which plays a critical role in preserving neuronal and retinal function. This imbalance coincided with retinal ...
journal_title:BioMed research international
pub_type: 杂志文章
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journal_title:BioMed research international
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journal_title:BioMed research international
pub_type: 杂志文章,评审
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pub_type: 临床试验,杂志文章
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pub_type: 杂志文章,已发布勘误
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pub_type: 杂志文章,评审
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更新日期:2018-07-19 00:00:00
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pub_type: 杂志文章
doi:10.1155/2020/8983494
更新日期:2020-10-16 00:00:00
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pub_type: 杂志文章
doi:10.1155/2015/705871
更新日期:2015-01-01 00:00:00
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pub_type: 杂志文章
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pub_type: 临床试验,杂志文章
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pub_type: 杂志文章,评审
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pub_type: 杂志文章,评审
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更新日期:2020-12-09 00:00:00
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pub_type: 杂志文章
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journal_title:BioMed research international
pub_type: 杂志文章,评审
doi:10.1155/2017/3691913
更新日期:2017-01-01 00:00:00
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journal_title:BioMed research international
pub_type: 杂志文章
doi:10.1155/2013/642848
更新日期:2013-01-01 00:00:00
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journal_title:BioMed research international
pub_type: 杂志文章
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journal_title:BioMed research international
pub_type: 临床试验,杂志文章
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journal_title:BioMed research international
pub_type: 杂志文章
doi:10.1155/2013/315848
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journal_title:BioMed research international
pub_type: 杂志文章,meta分析
doi:10.1155/2017/9892048
更新日期:2017-01-01 00:00:00