For example, proteins with small differences in molecular weight, such as the same protein with and without a posttranslational modification, tend to co-migrate during electrophoresis making them difficult to visualize simultaneously using chemiluminescence since the bands will most likely overlap. Overlapping bands can also impact detection of normalization and loading controls. Unless these controls are well resolved electrophoretically from the protein-of-interest, the blot must be either stripped and reprobed to detect the control, which renders the blot non-quantitative, or the controls must be placed on a separate blot, which is not a true loading control.
Because chemiluminescence relies on an enzyme-substrate reaction, the amount of signal (emitted light) is subject to variations in reaction kinetics, which can be affected by reaction conditions, i.e. pH, temperature, substrate concentration, and enzyme concentration. This inherent variability makes chemiluminescence, at best, a semi-quantitative detection chemistry.
The traditional use of x-ray film as a method of visualization suffers from dynamic range limitations of the film that can often lead to signal saturation. Using a digital imager can increase the linear dynamic range, allowing easier detection of low abundance proteins while limiting saturation when detecting high abundance proteins.