A Novel Gel Electrophoresis Technique 05SAR-PAGE:Invention,Application And Mechanism

Protein-protein interactions are fundamental to nearly all biological processes and the protein-protein interaction network characterization has become a research focus in the post-genomic era.In recent years,more and more attention has been paid to the study of weak interaction or oligosaccharide between proteins.The commonly used methods of protein interaction include analytical ultracentrifugation,nuclear magnetic resonance(NMR)and denaturation mass spectrometry,etc.These methods can obtain fine protein interaction information,but the operation process is relatively complicated and the costs are extremely expensive.Therefore,it is still a challenge to identify the weak interaction proteins with low cost technologies.Polyacrylamide gel electrophoresis(PAGE)is one of the most widely used technologies in biochemistry laboratories to identify the macromolecules and study their complexes.The most common gel electrophoresis method is SDS-PAGE,which can separate and identify proteins according to their molecular weight.But SDS-PAGE can not be used to study the weak interaction between proteins because SDS is a strong anionic surfactant,which destroys the structure of most proteins.Native-PAGE is a gel electrophoresis technology that can separate proteins without denaturing them.In Native-PAGE experiments,the migration rates of protein mixtures are related to the charge,size,and shape of proteins,therefore,the general protein marker may not accurately match the molecular weight of the intact protein.In order to solve these problems,05SAR-PAGE was invented to provide a convenient and inexpensive technique to analyze the proteins and identify weak protein interactions.The main research contents and conclusions are summarized as follows:Firstly,we found that there are two conformations of SAR in solution by NMR experiments.The binding of SAR to protein has no conformational selectivity,and the concentration of 0.05%w/v SAR has a mild effect on the structure of protein.Based on this,0.05%w/v SAR was used to replace the 0.1%w/v SDS in the traditional SDS-PAGE,and we established anew type of mild gel electrophoresis system 05SAR-PAGE.Through 05SAR-PAGE experiments on proteins with different molecular weights,it is found that proteins migrate according to molecular weight.The larger the relative molecular mass of protein,the more SAR binding,and the faster migration rate in gel electrophoresis.It shows that protein marker can be used to calibrate the molecular weight of proteins in 05 SAR-PAGE.Then,we used 05SAR-PAGE to separate the monomer and dimer protein states,analyze the acidic protein and the basic protein by the same protocol with the same protein marker.Monomer,homodimer,trimer and tetramer of Cyt c were identified by 05SAR-PAGE as well.We have demonstrated that 05SAR-PAGE can be used to separate the homodimer status of membrane protein CpxA.The dimerization of PhoBN in cell lysate can be directly detected by 05 SAR-PAGE and Western Blotting.Furthermore,un-and phosphorylated PhoBN were isolated and the monomer and dimer states of un-and methylated Cyt c were clearly detected by 05 SAR-PAGE.These results indicate that 05SAR-PAGE can be used to isolate and identify homologous oligomeric states of proteins with weak interaction and protein modification,with the advantages of simple operation,low price and wide application.Finally,we established a new two-dimensional electrophoresis method by combining 05 sar-page with SDS-PAGE,and successfully identified the membrane protein complex CpxA-OmpA in Escherichia coli cell membrane lysate.In summary,we have invented a new type of mild gel electrophoresis system 05SAR-PAGE,which is cheaper than SDS-PAGE.We have demonstrated that 05SAR-PAGE can be used to separate the monomer and dimer of proteins with weak interaction.05SAR-PAGE can be combined with Western blotting,and can be extended to two-dimensional level with other gel electrophoresis methods,such as SDS-PAGE.It is believed that the optimized two-dimensional 05SAR/SDS-PAGE will be applied to the identification of protein complexes in more complicated conditions.