Effect of salt concentration on separation patterns in static capillary isoelectric focusing with imaging detection.

Salts introduced into protein samples have an impact on the pH gradient in free solution in isoelectric focusing (IEF), which is reflected by the separation pattern. In this study, samples containing different concentrations of phosphate-buffered saline (PBS) were focused in capillary format and detected in a real-time mode using an imaged capillary isoelectric focusing (CIEF) system at 280 nm. It was observed that salt compressed the pH gradient with a degree of 4.3% at a PBS concentration interval of 10 mM. As a result, the same sample components, therefore, were focused at different positions inside the capillary. Using two pI markers as the internal standards, the separation patterns in the presence of salts were corrected to the salt-free matrix by simply stretching the electropherograms. The stretched electropherograms of model samples, pI markers and myoglobin, demonstrated the feasibility of this correction. This simple method is promising for identifying proteins, which may exhibit different pI values after their mutation and stability process, when salt is present in the sample.