Capillary isoelectric focusing on the Agilent capillary electrophoresis system service manual说明书用户手册.PDF

Capillary isoelectric focusing on the Agilent capillary electrophoresis system Technical Note Introduction The cIEF method described here capillary and the detection point This Technical Note describes the is a two step method. First, the so that focusing of the proteins setup of a capillary isoelectric capillary is filled with a mixture takes place between the capillary focusing (cIEF) method to run on of protein sample and ampholytes inlet and detection point. This an Agilent capillary electrophore- which is then focused by applying enables the detection of very basic sis (CE) system. With this method a high field strength (V/cm). The proteins. As a starting point for pI proteins and peptides can be sepa- ampholytes form a pH gradient in determination, we recommend a rated according to differences in the capillary and the proteins are broad range of ampholytes cover- their isoelectric points (pI). focused into sharp zones at the ing pI values from 3 to 10 pH region which corresponds to (figure 1). The pI of a protein is an important their pI. In the second step the characteristic and cIEF can be contents of the capillary are used to either determine a pro- mobilized past the detector by tein’s pI or to perform separations applying pressure to the inlet vial based on pI differences1. Separa- while maintaining the field tions based on pI are used in pro- strength. Focused peaks are mon- tein analysis for identification, itored by the detector and appear purity assessment, degradation in order from basic to acidic pro- checking, microheterogeneity teins. TEMED is use