Westerberg Leo J S, Dedic Benjamin, Näslund Erik, Thorell Anders, Spalding Kirsty L
Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
Department of Clinical Sciences, Danderyd Hospital, Karolinska Institutet, Stockholm, Sweden.
PLoS One. 2025 Jul 22;20(7):e0328136. doi: 10.1371/journal.pone.0328136. eCollection 2025.
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.
自20世纪70年代末以来，蛋白质印迹法一直是分析蛋白质表达的关键方法，但对于最佳归一化策略尚未达成既定共识。在本研究中，使用原代成熟人类脂肪细胞来研究管家蛋白和总蛋白（TP）作为蛋白质印迹法归一化参考的稳健性。与所有研究的管家蛋白相比，TP在技术重复中表现出最低的变异性，并且是所选目标蛋白的优质归一化参考。当以蛋白质梯度加载时，TP也显示出与预期值最接近的一致性，突出了TP作为归一化标准的动态强度。此外，与在三个代谢相似个体中研究的管家蛋白相比，TP在个体内和个体间始终表现出较低的变异性。总之，TP归一化是原代成熟人类脂肪细胞中可靠蛋白质表达分析的首选方法。
