Capillary isoelectric focusing coupled with dynamic imaging detection: A one-dimensional separation for two-dimensional protein characterization

Here, we devised a novel approach for two-dimensional (2D) protein characterization using a single one-dimensional separation followed by a second characterization in the same instrument. The approach combines capillary isoelectric focusing (CIEF), which separates proteins according to pI, with dynamic imaging detection, which permits monitoring of protein diffusion in real time and thereby allows estimation of molecular weight from diffusion coefficient. Compared with classical 2D gel electrophoresis, the approach provided several significant advantages including speed and ease in operation and automation, while yielding comparable accuracy. The approach was applicable for protein samples of low to moderate complexity.