Protein Preparative Isolation By An Innovative Electroeluter Coupled With Polyacrylamide Gel

Polyacrylamide gel electrophoresis(PAGE)is an important tool for qualitative and quantitative analysis of protein.It has been used for preparative isolation of many kinds of biomolecules due to its advantages,such as high resolution,low cost and easy to control.The prepared proteins from PAGE can be used in various downstream applications,such as protein chemistry,amino acid composition determination and antigen identification etc.So how to recover proteins from PAGE is concerned by scientists.Up to now,three main methods(passive elution,gel dissolving and electroelution)are recommended.For the commonly used electroelution method,target protein jailed in gel was driven into surrounding elution buffer under electric field force.Experimental conditions of electroelution are mild and not involved in any harsh detergent.However,there are still several shortages in conventional electroelution,such as complex operation,time-consuming,low recovery rate and diluted protein sample,which needs further concentration steps.Herein,in order to solve the above problems,a novel small electroelution device,ring-shaped electroeluter(RSE),was designed and manufactured for the recovery of high concentration protein from PAGE.The principle work of this study includes three chapters.(1)Design and manufacture of the RSE and validation of feasibility.Based on the previous electroelution device designed by our laboratory,a novel RSE was designed by Multiphysics software and manufactured via CNC milling machine.The RSE device mainly consists of two parts,the upper cathode and lower anode.The cathode part is a cylinder with a ring-shaped well used for placing the protein band of interest and a small enrichment well in the center.The enrichment well with a pore at the bottom is separated with dialysis membrane to collect the eluted proteins.The enrichment well is immersed into the tank of the cylindrical anode.Under the force of electric field,the target proteins migrates from gel into the elution buffer,and then keep moving into enrichment well.Then model protein was used to evaluate the newly designed device compared with conventional electroelution.The results demonstrate that RSE has many advantages,such as short elution time,higher concentration of protein sample and higher recovery etc.(2)Preparative isolation of HSA from serum via RSE coupled with SDS-PAGE.In order to verify that RSE can be used for preparative isolation of protein from real sample,serum was used as model real sample and was separated by SDS-PAGE.Then RSE was used to recover target protein.First,the experimental conditions for running RSE were optimized,including voltage and elution time.Under the optimized conditions,HSA was successfully isolated from serum,meaning the newly designed RSE has potential application in preparative isolation of protein from real sample based on molecular weight coupled with SDS-PAGE.(3)Preparative isolation of the biomarker of diabetes(HbA1c)from diabetes via RSE coupled with IPG-IEF-PAGE.In order to verify that RSE can be used for preparative isolation of proteins based on the pI,blood of diabetes was used as model real sample and was separated by IPG-IEF-PAGE.Then RSE was used to recover HbA1 c.First,the experimental conditions for running IPG-IEF-PAGE was optimized,including pH range,running time and voltage.Under optimized conditions(pH range is 6-9),HbA1 c was successfully isolated,indicating that RSE has potential in preparative isolating biomarker from complex sample based on protein pI coupled with IPG-IEF-PAGE.