cIEF is a method used to run on the Agilent Capillary Electrophoresis (CE) system. It can separate proteins and peptides according to differences in their isoelectric points (pI). The pI of a protein is an important characteristic and cIEF can be used to either determine a protein's pI or to perform separations based on pI differences. Separations based on pI are used in protein analysis for identification, purity assessment, degradation checking, microheterogeneity analysis, clinical diagnosis, and the analysis of complex protein mixtures. The cIEF method described here is a two step method. First, the capillary is filled with a mixture of protein sample and ampholytes which is then focused by applying a high field strength (V/cm). The ampholytes form a pH gradient in the capillary and the proteins are focused into sharp zones at the pH region which corresponds to their pI. In the second step the contents of the capillary are mobilized past the detector by apply- ing pressure to the inlet vial while maintaining the field strength. Focused peaks are monitored by the detector and appear in order from basic to acidic proteins. TEMED is used as a spacer between the outlet end of the capillary and the detec- tion point so that focusing of the proteins takes place between the capillary inlet and detectio
The document details a specific protocol for Capillary Isoelectric Focusing (cIEF) designed to run on the Agilent Capillary Electrophoresis system. This two-step method first focuses protein samples into sharp zones at their isoelectric point (pI) using ampholytes and high field strength, then mobilizes them past a UV detector for analysis. The technique allows for the determination of pI values or separation based on pI differences, which is useful for protein identification, purity assessment, and clinical diagnosis.
The document recommends using a protein concentration range from 0.5 to 2.5 mg/mL.
Decreasing the mobilization pressure from 50 to 25 mbar significantly improves pl resolution, however, with a longer run time.