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 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.