Construction Of Multi-Compartment Electrophoresis Separation Systen And Application In Separation Of Protein And Small Molecule

It is very important to study of the interaction between protein and small molecule, for pharmacology, drug development, biomarker identification, clinical diagnosis, proteomics and metabolomics. However, the separation of proteins and small molecules plays an important role in the study of interaction between protein and small molecule. But, the existing methods have some disadvantages, such as time-consuming, laborious. In this study, a novel electrophoresis separation system was constructed, it could achieve separation of protein and small molecule, the multi-compartment electrophoresis separation system. The separation system can be used in the study of interaction between proteins and small molecules in the process of separation of proteins and small molecules. This thesis introduces the constitution of multi-compartment, namely the circulatory system, separating system, cooling system and power system. Meanwhile, the various factors affecting the separation effect of electrophoresis were optimized, including: voltage, electrophoresis time, buffer concentration, membrane choice etc.. When the applied8V voltage, electrophoresis time15min (5min/each),10mM phosphate buffer solution buffer solution (pH=7.2), membrane was0.2μm cellulose acetate membrane and10KD polyethersulfone ultrafiltration membranes, we obtained the best separation conditions. Under the optimal conditions,90%more protein moved to the acceptor compartment from the donor compartment and separated with free neutral small molecules in the electric field force. Then we use the reversed phase high performance liquid chromatography for detecting the protein and neutral small molecules. The experimental results show that the electrophoresis separation system was effective to the separation of proteins and small neutral molecules. In this thesis, the separation system was evaluated, separation of protein was72.53%, relative standard deviation (RSD%) ranged below than13.75%.The experimental results show that the method is simple, rapid and easy to operate. Moreover, it has big potential to study of protein and small molecule interactions, protein screening, drug screening and has a good application prospect.