Fig 1.
Impact of BSA contamination on protein quantification methods.
(A) Stain-free image of total protein (TP) with bands corresponding to 50 and 75 kDa indicated on ladder. Duplicates shown for each condition. (B) BSA (65 kDa), tubulin (55 kDa) and GLUT4 (54 kDa) staining. (C) Average BSA signal from all duplicates, normalized to the smallest value above 0. (D) Average tubulin signal from all duplicates, normalized to the smallest value. (E) Average BSA signal from all duplicates, normalized to tubulin, normalized to smallest value above 0. (F) Tubulin normalized to TP, normalized to the smallest value.
Fig 2.
Technical replicates from OBNI individuals.
Coefficient of variation (CV) for total protein (TP) and respective housekeeping proteins, normalized to the sum (left panels), and for GLUT4 normalized to TP or housekeeping proteins (right panels). Each dot represents one well – a technical replicate of the same lysate sample run in separate lanes. TP demonstrated the lowest CV across all technical replicate blots, except when compared to actin in the OM sample (A–F). When normalizing GLUT4, TP showed the lowest CV in four blots (A, B, E, and F), while actin (C) and ENOA (D) exhibited lower CVs in two blots.
Fig 3.
OBHI OM lysate loaded in 2.5 µg gradient increments (2.5–20 µg).
Lysates were visualized with a color gradient, and the coefficient of variation (CV) was calculated. Total protein (TP) showed the lowest CV across all blots when normalized to the sum (A–C). For GLUT4 normalization, PARK7 demonstrated the lowest CV in one blot (D), while TP achieved the lowest CV in two blots (E and F).
Fig 4.
Protein gradient loading across a metabolically and clinically similar cohort (n = 3).
Lysates were loaded at 5, 10, and 15 µg, visualized with a color gradient, and normalized to the minimum value. Inter- and intra-individual comparisons of normalization references and GLUT4 demonstrated that total protein (TP) exhibited the lowest variability both within and between individuals (A–C). In contrast, housekeeping proteins normalized to TP reveal substantial inter-individual variability (D–F), highlighting that their expression levels differ between patients even when loading is accounted for. GLUT4 normalized to different references (TP or housekeeping proteins) further illustrate how the choice of normalization strategy can markedly influence the apparent between-patient differences (G–I).
Table 1.
Optimal normalization references for different experimental designs.