Proteins Analysis Based On Capillary Isoelectric Focusing

The isoelectric point(pI) is one of the most important physicochemical parameters of amphoteric biological particles, such as proteins, polypeptides, microorganisms, cells and organelles. The capillary isoelectric focusing(cIEF) is the method to determine the pI of amphoteric substances, which is based the technology of capillary electrophoresis. cIEF can not only be used to characterize proteins and peptides with different pI, but also to separate and detect multiple sample components. Moreover, the study of intermolecular interactions can aslo be accomplished by cIEF. The laser-induced fluorescence(LIF) detection is one highly sensitive detection technology, which has the advantages of low detection limit, short detection time, low sample consumption and online testing. The cIEF coupled with LIF detection also has the advantages of high efficiency, speed and sensitivity.This paper includes the following parts:(1) The one-step method and two-step method of cIEF were compared. The improvement of one-step method of cIEF was accomplished by optimizing experiment parameters of solution composition, injection volume, focusing voltage and pH of electrode solution, etc. The results indicated that the improved one-step method had the advantages of fast separation, complete focusing, simple process and extensive adaptability.(2) The improved one-step cIEF was applied to the analysis and detection of multiple fusion proteins and satisfying resolution and linearity was got. The protein’s pI detected by cIEF had good consistency with the theory value provided by protein’s manufacture factory, which indicated that the one-step cIEF could be applied in the separation and purification, quality control, stability evaluation and pI determination of practical samples.(3) Both cIEF-LIF and CZE-LIF were used for the analysis of green fluorescent protein(GFP). Experiment parameters, such as separation voltage, buffer pH, electrode solution pH, were optimized. And those laid the foundation for the further study of interactions between GFP( and other fluorescent proteins) and nucleic acids, as well as the screening of aptamers of(GFP).