Titration-WB: A methodology for accurate quantitative protein determination overcoming reproducibility errors.
Western blotting (WB) is a cornerstone technique for protein detection and quantification in molecular biology. However, its semi-quantitative nature, reliance on housekeeping protein normalization, and susceptibility to technical variability often undermine data accuracy and reproducibility. To add...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2025-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0325052 |
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| author | Alice Maestri Ewa Ehrenborg Olivera Werngren Maria Olin Carolina E Hagberg Matteo Pedrelli Paolo Parini |
| author_facet | Alice Maestri Ewa Ehrenborg Olivera Werngren Maria Olin Carolina E Hagberg Matteo Pedrelli Paolo Parini |
| author_sort | Alice Maestri |
| collection | DOAJ |
| description | Western blotting (WB) is a cornerstone technique for protein detection and quantification in molecular biology. However, its semi-quantitative nature, reliance on housekeeping protein normalization, and susceptibility to technical variability often undermine data accuracy and reproducibility. To address these limitations, we introduce titration-based Western blotting (t-WB), as innovative quantitative approach that uses serial dilutions of protein samples to generate regression curves for precise protein quantification. The method mitigates common errors, including loading inaccuracies and signal saturation, by leveraging the R² value as a quality control metric and calculating the regression line. Its slope is then used as a measure of protein concentration, expressed as signal intensity/total protein loaded. The advantage of t-WB is the removal of housekeeping protein normalization, eliminating thus the bias created by experimental conditions that may alter housekeeping protein levels. t-WB was validated across diverse setups, demonstrating its robustness in minimizing inter-experiment variability and improving accuracy by normalizing to a single internal control. By standardizing workflows, t-WB ensures reproducibility, uncovers subtle biological changes, and resolves biases inherent to classical WB protocols. |
| format | Article |
| id | doaj-art-3784be2262004b41a5e460a57915149a |
| institution | DOAJ |
| issn | 1932-6203 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-3784be2262004b41a5e460a57915149a2025-08-20T03:20:09ZengPublic Library of Science (PLoS)PLoS ONE1932-62032025-01-01206e032505210.1371/journal.pone.0325052Titration-WB: A methodology for accurate quantitative protein determination overcoming reproducibility errors.Alice MaestriEwa EhrenborgOlivera WerngrenMaria OlinCarolina E HagbergMatteo PedrelliPaolo PariniWestern blotting (WB) is a cornerstone technique for protein detection and quantification in molecular biology. However, its semi-quantitative nature, reliance on housekeeping protein normalization, and susceptibility to technical variability often undermine data accuracy and reproducibility. To address these limitations, we introduce titration-based Western blotting (t-WB), as innovative quantitative approach that uses serial dilutions of protein samples to generate regression curves for precise protein quantification. The method mitigates common errors, including loading inaccuracies and signal saturation, by leveraging the R² value as a quality control metric and calculating the regression line. Its slope is then used as a measure of protein concentration, expressed as signal intensity/total protein loaded. The advantage of t-WB is the removal of housekeeping protein normalization, eliminating thus the bias created by experimental conditions that may alter housekeeping protein levels. t-WB was validated across diverse setups, demonstrating its robustness in minimizing inter-experiment variability and improving accuracy by normalizing to a single internal control. By standardizing workflows, t-WB ensures reproducibility, uncovers subtle biological changes, and resolves biases inherent to classical WB protocols.https://doi.org/10.1371/journal.pone.0325052 |
| spellingShingle | Alice Maestri Ewa Ehrenborg Olivera Werngren Maria Olin Carolina E Hagberg Matteo Pedrelli Paolo Parini Titration-WB: A methodology for accurate quantitative protein determination overcoming reproducibility errors. PLoS ONE |
| title | Titration-WB: A methodology for accurate quantitative protein determination overcoming reproducibility errors. |
| title_full | Titration-WB: A methodology for accurate quantitative protein determination overcoming reproducibility errors. |
| title_fullStr | Titration-WB: A methodology for accurate quantitative protein determination overcoming reproducibility errors. |
| title_full_unstemmed | Titration-WB: A methodology for accurate quantitative protein determination overcoming reproducibility errors. |
| title_short | Titration-WB: A methodology for accurate quantitative protein determination overcoming reproducibility errors. |
| title_sort | titration wb a methodology for accurate quantitative protein determination overcoming reproducibility errors |
| url | https://doi.org/10.1371/journal.pone.0325052 |
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