Why native gel electrophoresis doesn’t separate effectively by molecular weight
Native gel electrophoresis does not separate effectively by molecular weight because the native gel electrophoresis technique does not use involve denaturation of the proteins. The proteins retain their folded structure and migrate through the gel according to their mass:charge ratio. The charge of a protein is based on the ionizable side groups of its amino acids. This means that two different proteins with similar molecular weights may travel different distances due to different charges. Using sodium dodecyl sulfate (SDS) in denaturing polyacrylamide gel electrophoresis(PAGE) overcomes this problem because the SDS molecules bind to the protein chains in a constant ratio of about 1.4g of SDS per gram of protein and overcome the charges of the ionizable side groups. This makes the net charge of all the proteins negative so that all the proteins migrate toward the positive anode when the electric current is applied. SDS also changes the native conformation of the proteins into a constant rod-like conformation. This allows the protein chains to move according to their primary structure, and eliminates the differences in shapes that are found in the native secondary and tertiary structures. This allows separation of the proteins based predominantly on molecular mass.
