A critical path to producing high quality, reproducible data from quantitative western blot experiments
Abstract Western blotting experiments were initially performed to detect a target protein in a complex biological sample and more recently, to measure relative protein abundance. Chemiluminescence coupled with film-based detection was traditionally the gold standard for western blotting but accurate...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-10-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-22294-x |
| _version_ | 1828318077440229376 |
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| author | Sean C. Taylor Luciana K. Rosselli-Murai Bélinda Crobeddu Isabelle Plante |
| author_facet | Sean C. Taylor Luciana K. Rosselli-Murai Bélinda Crobeddu Isabelle Plante |
| author_sort | Sean C. Taylor |
| collection | DOAJ |
| description | Abstract Western blotting experiments were initially performed to detect a target protein in a complex biological sample and more recently, to measure relative protein abundance. Chemiluminescence coupled with film-based detection was traditionally the gold standard for western blotting but accurate and reproducible quantification has been a major challenge from this methodology. The development of sensitive, camera-based detection technologies coupled with an updated technical approach permits the production of reproducible, quantitative data. Fluorescence reagent and detection solutions are the latest innovation in western blotting but there remains questions and debate concerning their relative sensitivity and dynamic range versus chemiluminescence. A methodology to optimize and produce excellent, quantitative western blot results with rigorous data analysis from membranes probed with both fluorescent and chemiluminescent antibodies is described. The data reveal when and how to apply these detection methods to achieve reproducible data with a stepwise approach to data processing for quantitative analysis. |
| first_indexed | 2024-04-13T17:36:54Z |
| format | Article |
| id | doaj.art-5eb803a0e2b9413991b9075c7b1620f9 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-13T17:36:54Z |
| publishDate | 2022-10-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-5eb803a0e2b9413991b9075c7b1620f92022-12-22T02:37:19ZengNature PortfolioScientific Reports2045-23222022-10-0112111210.1038/s41598-022-22294-xA critical path to producing high quality, reproducible data from quantitative western blot experimentsSean C. Taylor0Luciana K. Rosselli-Murai1Bélinda Crobeddu2Isabelle Plante3GENSCRIPT USA INCGENSCRIPT USA INCInstitut National de la Recherche Scientifique - Armand-Frappier Santé BiotechnologieInstitut National de la Recherche Scientifique - Armand-Frappier Santé BiotechnologieAbstract Western blotting experiments were initially performed to detect a target protein in a complex biological sample and more recently, to measure relative protein abundance. Chemiluminescence coupled with film-based detection was traditionally the gold standard for western blotting but accurate and reproducible quantification has been a major challenge from this methodology. The development of sensitive, camera-based detection technologies coupled with an updated technical approach permits the production of reproducible, quantitative data. Fluorescence reagent and detection solutions are the latest innovation in western blotting but there remains questions and debate concerning their relative sensitivity and dynamic range versus chemiluminescence. A methodology to optimize and produce excellent, quantitative western blot results with rigorous data analysis from membranes probed with both fluorescent and chemiluminescent antibodies is described. The data reveal when and how to apply these detection methods to achieve reproducible data with a stepwise approach to data processing for quantitative analysis.https://doi.org/10.1038/s41598-022-22294-x |
| spellingShingle | Sean C. Taylor Luciana K. Rosselli-Murai Bélinda Crobeddu Isabelle Plante A critical path to producing high quality, reproducible data from quantitative western blot experiments Scientific Reports |
| title | A critical path to producing high quality, reproducible data from quantitative western blot experiments |
| title_full | A critical path to producing high quality, reproducible data from quantitative western blot experiments |
| title_fullStr | A critical path to producing high quality, reproducible data from quantitative western blot experiments |
| title_full_unstemmed | A critical path to producing high quality, reproducible data from quantitative western blot experiments |
| title_short | A critical path to producing high quality, reproducible data from quantitative western blot experiments |
| title_sort | critical path to producing high quality reproducible data from quantitative western blot experiments |
| url | https://doi.org/10.1038/s41598-022-22294-x |
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