Fig. 3

Assessing the Resolution of the Method. (A) Calibration curves of the same icIEF experiment. Certified values of ten pI markers (5.85, 6.14, 7.05, 7.60, 8.18, 8.40, 9.00, 9.50, 10.00, 10.17) are plotted against their positions along the capillary. Linear regression (blue dotted line: using only 5.85, 9.99, 10.17 external pI markers) and non-linear regression (6th-order polynomial, red line, using all pI markers) were applied. (B) Method resolution along the pH gradient, corresponding to A. This graph shows the distance (pixels) between different points of the pH gradient, spanning a ΔpH of 0.2 units. Resolution values were derived from the regressions in A. Notably, linear calibration assumes constant resolution, while non-linear regression allows resolution to vary along the gradient. (C) Calibration curves for two different pI marker sets: ProteinSimple (4.05, 5.85, 7.05, 9.50, 9.99—orange) and SCIEX (4.10, 5.50, 7.00, 9.50, 10.00—blue) diluted in the same parent solution. (D) Resolution corresponding to the marker sets in C. Using the same parent solution highlights that differences in resolution curves are due to solvent effects of the two pI marker formulations, significantly influencing the pH gradient. (E) Ten pI markers (5.85, 6.14, 7.00, 7.60, 8.18, 8.40, 9.00, 9.50, 10.00, 10.17) run in icIEF with different ampholyte stratifications (4% 3–10—black line; 6% 1:1 3–10/8–10.5—red line; 4% 1:1 5–8/8–10.5—blue line). (F) As shown by the calibration curves in E, ampholyte composition strongly impacts resolution along the pH gradient. However, these variations are difficult to predict without systematic experimental design.