Superior normalization using total protein for western blot analysis of human adipocytes.
Western blotting has been a pivotal method for analyzing protein expression since the late 1970s, yet there is no established consensus on an optimal normalization strategy. In this study, primary mature human adipocytes were used to investigate the robustness of housekeeping proteins and total prot...
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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.0328136 |
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| author | Leo J S Westerberg Benjamin Dedic Erik Näslund Anders Thorell Kirsty L Spalding |
| author_facet | Leo J S Westerberg Benjamin Dedic Erik Näslund Anders Thorell Kirsty L Spalding |
| author_sort | Leo J S Westerberg |
| collection | DOAJ |
| description | Western blotting has been a pivotal method for analyzing protein expression since the late 1970s, yet there is no established consensus on an optimal normalization strategy. In this study, primary mature human adipocytes were used to investigate the robustness of housekeeping proteins and total protein (TP) as normalization references for western blotting. TP exhibited the lowest variance among technical replicates compared to all investigated housekeeping proteins and was a superior normalization reference for the chosen protein-of-interest. TP also demonstrated the closest alignment with expected values when loaded as a protein gradient, highlighting the dynamic strength of TP as a normalization standard. Additionally, TP consistently demonstrated lower intra- and inter-individual variability in comparison to housekeeping proteins investigated across three metabolically similar individuals. In conclusion, TP normalization is the preferred method for reliable protein expression analysis in primary mature human adipocytes. |
| format | Article |
| id | doaj-art-d431a13569644e7bb26d5e8dd68d86fa |
| institution | Kabale University |
| issn | 1932-6203 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-d431a13569644e7bb26d5e8dd68d86fa2025-08-20T03:32:04ZengPublic Library of Science (PLoS)PLoS ONE1932-62032025-01-01207e032813610.1371/journal.pone.0328136Superior normalization using total protein for western blot analysis of human adipocytes.Leo J S WesterbergBenjamin DedicErik NäslundAnders ThorellKirsty L SpaldingWestern blotting has been a pivotal method for analyzing protein expression since the late 1970s, yet there is no established consensus on an optimal normalization strategy. In this study, primary mature human adipocytes were used to investigate the robustness of housekeeping proteins and total protein (TP) as normalization references for western blotting. TP exhibited the lowest variance among technical replicates compared to all investigated housekeeping proteins and was a superior normalization reference for the chosen protein-of-interest. TP also demonstrated the closest alignment with expected values when loaded as a protein gradient, highlighting the dynamic strength of TP as a normalization standard. Additionally, TP consistently demonstrated lower intra- and inter-individual variability in comparison to housekeeping proteins investigated across three metabolically similar individuals. In conclusion, TP normalization is the preferred method for reliable protein expression analysis in primary mature human adipocytes.https://doi.org/10.1371/journal.pone.0328136 |
| spellingShingle | Leo J S Westerberg Benjamin Dedic Erik Näslund Anders Thorell Kirsty L Spalding Superior normalization using total protein for western blot analysis of human adipocytes. PLoS ONE |
| title | Superior normalization using total protein for western blot analysis of human adipocytes. |
| title_full | Superior normalization using total protein for western blot analysis of human adipocytes. |
| title_fullStr | Superior normalization using total protein for western blot analysis of human adipocytes. |
| title_full_unstemmed | Superior normalization using total protein for western blot analysis of human adipocytes. |
| title_short | Superior normalization using total protein for western blot analysis of human adipocytes. |
| title_sort | superior normalization using total protein for western blot analysis of human adipocytes |
| url | https://doi.org/10.1371/journal.pone.0328136 |
| work_keys_str_mv | AT leojswesterberg superiornormalizationusingtotalproteinforwesternblotanalysisofhumanadipocytes AT benjamindedic superiornormalizationusingtotalproteinforwesternblotanalysisofhumanadipocytes AT eriknaslund superiornormalizationusingtotalproteinforwesternblotanalysisofhumanadipocytes AT andersthorell superiornormalizationusingtotalproteinforwesternblotanalysisofhumanadipocytes AT kirstylspalding superiornormalizationusingtotalproteinforwesternblotanalysisofhumanadipocytes |