EDVOTEK® BIOTECHNOLOGY BOOTCAMP
Proteins and SDS-PAGE
SDS polyacrylamide-gel electrophoresis, or SDS-PAGE, is a technique that is used to separate proteins according to their molecular weight. Proteins produce a unique challenge for electrophoresis because they have complex shapes and different charges, which affect how they migrate through the gel. In order to accurately separate proteins by molecular weight and not by shape or charge, the secondary structure of the protein is unfolded using the anionic detergent sodium dodecyl sulfate (SDS) and a reducing agent. The SDS molecules form a complex with the protein, negating its inherent charge. The reducing agent breaks covalent bonds that link protein subunits.
After denaturation, the mixture of proteins is added into depressions (or “wells”) within a gel, and then an electrical current is passed through the gel. Because the SDS-protein complex has a strong negative charge, the current drives the proteins through the gel towards the positive electrode. At first glance, a polyacrylamide gel appears to be a solid. On the molecular level, the gel contains channels through which the proteins can pass. Small proteins move through these holes easily, but large proteins have a more difficult time squeez- ing through the tunnels. Because molecules of different sizes travel at different speeds, they separate into discrete “bands” within the gel. After the current is stopped, the bands are visualized using a stain that sticks to proteins.
PROTEIN EXPERIMENTS:
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