Enhancement of resolution in two‐dimensional gel electrophoresis and simultaneous resolution of acidic and basic proteins

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Abstract
In studies of genetic diseases such as Duchenne muscular dystrophy (DMD), two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) provides an indispensable tool for studying protein changes. For optimum use of 2‐D PAGE it is important to maximize the resolution that can be obtained. In this study we have developed a system using flat‐bed isoelectric focusing (IEF) with gels bound to a plastic backing for the first‐dimension separation. This system, when compared to the conventional rod IEF system, was found to yield good resolution of both the acidic and basic proteins, which are normally separated using non‐equilibrium systems. We have also used the principals of gradient engineering in both the IEF and sodium dodecyl sulfate (SDS)‐PAGE dimensions to maximize the gel area used for the separations. Mixtures of various commercial carrier ampholytes were also used and were found to further enhance resolution.