Principle and Protocol of SDS-PAGE
PrincipleThe concentration of polyacrylamide gels can be prepared as required in two electrophoresis systems —called “continuous system” and “discontinuous system”. The biggest feature of “discontinuous system” lies in its greatly improved sample separation resolution. Main features of this electrophoresis are: (1) Use of two gel systems with different concentrations; (2) Solution composition and pH are different for the preparation of the two gel and are also different from electrophoresis buffer composition and pH in electrophoresis tank. In the experiment, electrophoresis gel is divided into two layers: the upper one is a macroporous gel with low concentration, called stacking gel, buffer for the formulation of this layer is Tris-HCl, pH6.7; the lower one is hole glue with high concentrations, called separating gel or electrophoresis gel , and the buffer for this is Tris-HCl, pH8.9. Electrode buffer in the electrophoresis tank is Tris- glycine, pH8.3. Obviously, the gel concentrations, compositions, pH and the electrophoresis buffer systems are different from each other, thus forming a discontinuous system.
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