Polyacrylamide Gel Electrophoresis Based On Moving Reaction Boundary And Study Of Its Application

As a classical technology for separation and analysis, polyacrylamidegel electrophoresis (PAGE) has been widely used to separate and analyzeproteins, RNAs and nanoparticles in many biological, agricultural, chemicalor medical labs due to its mature operation, fascinating separation effect andcommercial equipment. Since it was advanced in1960s, the PAGE hasdeveloped in both its application scope and method sensitivity. However,there are still two problems. Firstly, most of the PAGE methods are based onisotachophoresis (unlike more than ten stacking techniques in capillaryelectrophoresis). Secondly, the volume of sample is generally smaller than15l in the classic ITP-PAGE because of poor condensation efficiency in large volume loading. In recent years, the theory of moving reactionboundary develops very fast, especially for moving neutralization boundary,which has been used for fundamental study of isoelectric focusing, acid-basetitration and application study of the ampholytes and small molecule drugsstacking. This study applied the theory of MRB to PAGE, solved the twoexisting problems, separated and stacked hemoglobins (as a model protein)successfully and increased the sensitivity at least eight-fold.There are three parts in the review.1. The basic knowledge of PAGE, itsdevelopment and applications.2. The development, classification andapplication of the principle used in the new system, moving reactionboundary.3. Species and properties of hemoglobins, implication andanalytical methods of the two key heomglobins related to clinical diagnoses.In the experimental section, model of MRB-PAGE was designed firstly andseparation and identification was done via the combination of isoelectricfocusing and MALDI-TOF MS.In the first section of experiments, we set up the theory model ofMRB-PAGE to separate neutral proteins based on the exsiting commercialvertical electrophoresis device and optimized the parameters, such asconcentration of the gel, degree of crosslinking and pH value andconcentration of the three buffers. In the second section, we used the optimized system to separate the hemoglobins and compared the results withthat of other models including the classic PAGE, continuous PAGE andnon-continuous PAGE. The outcome was that the sensitivity improved atleast eight fold and satisfied identification results were achieved by the twomethods (IEF and MS).The whole experiment proved MRB-PAGE could increase thesensitivity and resolution of large-volume low-abundance protein sample.Thus, the study provides guidance to development of online sample stackingin slab gel electrophoresis, and the simple method developed is of greatpotential use in separation and analysis of diluted protein sample inbiomedical fields.