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Post Electrophoretic Analysis Articles

The Polyacrylamide Matrix-Buffer Strength

The buffer system in electrophoresis controls the pH of the gel, preventing damage to sample molecules and, in certain cases, controlling the ionization state of the molecules. A second, though no less significant function, derives from the fact that the vast majority of current flowing through the electrophoresis gel is carried by the buffer ions. For a homogeneous system like denaturing PAGE electrophoresis of DNA, in which the type and concentration of buffers in the tank and gel are the same, the buffer prevents wide swings in pH and controls the conductivity of the gel. For thenative electrophoresis of proteins, the buffer pH has the added function of controlling the state of ionization of the samples. In this second case, even slight changes in pH can result in large effects on the relative mobility of sample components. In a multiphasic system, such as SDS-PAGE electrophoresis of proteins, where buffers in the tank and gel are different, the considerations of buffer design can take on an even greater level of complexity.

In all cases, the ionic strength of the buffer in the gel must be sufficient to keep the sample in solution and to provide sufficient buffering capacity. Higher concentrations of gel buffer will also slow the diffusion of samples, and result in sharper bands. The benefits of higher buffer concentrations must, however, be balanced against the fact that the more concentrated the buffer in the gel, the higher the electrical conductivity. With higher concentration, at a given voltage, the current will be greater and more heat will be generated. Because of the problems caused by excessive heating, high buffer concentrations must be accompanied by a low voltage gradient.

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