Research Of Substitute Material Of Polyacrylamide Gel With Low Toxicity

Polyacrylamide gel electrophoresis is widely used in the separation and identification of protein and polypeptide in many fields such as biology, medicine, food, agriculture, depending on its merits of low cost, simple operation, good repetition, shorter analysis time. The mechanism of separation of polyacrylamide gel electrophoresis is that protein would migrate in the net structure of polyacrylamide under application of an electric field, and the moving distance has a close relationship to electrophoretic conditions as well as molecular weight, shape and quantity of electric charge of a protein. While ionic detergent (SDS) and strong reducing agent (?-mercaptoethanol) are added to electrophoretic buffer solution and protein buffer solution separately in SDS-PAGE, the migration distance of a protein is mainly determined by molecular weight, regardless of the effects of charge factors.Acrylamide monomer is needed when in the process of gel preparation, and acrylamide would polymerize under the catalysis of chain initiator and accelerator, to form a stable solid gel. Acrylamide monomer is powder in normal temperature and pressure, and it could enter the body through the skin, the digestive tract, the respiratory tract easily when preparing a solution. A small amount of acrylamide is still in the gel in the form of monomer when the solid gel is formed. Even more important, acrylamide also has potential reproductive toxicity and neurotoxicity, and skin allergies may occur even a small amount of contact with the solution. Therefore, we are looking forward to a substitution of acrylamide, which has a lower toxicity than acrylamide and could keep polyacrylamide gel separation advantage.I selected two compounds containing amide structure: N,N-dimethylacrylamide and N-(2-hydroxyethyl)acrylamide in my experiment. Firstly, I optimized the addition amount of accelerator TEMED to the system, and the addition of TEMED in 5mL 10% separation gel is 12.5?L and in 2mL 5% separation gel is 5?L in both two gels; secondly, I optimized the ratio of amide monomer and crosslinker (Bis). The ratio of monomer and crosslinker is 29:0.8, could make the aperture size of the gel appropriate for protein separation. Next, the gel for experimental use was prepared according to the optimized conditions and I detected the protein(BSA) in Western Blot technique. PHEA gel can be uesd in transformation and antigen-antibody reaction would happen on membrane.Finally, the perfusion gradient gel was made with the help of the peristaltic pump, which could adjust the flow velocity of different components, and enhance the resolution of proteins with different molecular weight, to make gel preparation process automatic and reduce the burden of human.