Two-Dimensional PAGE Using Flat-Bed IEF in the First Dimension
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Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) of proteins involves the use of two independent separation techniques resulting in the ability to resolve very complex mixtures. Normally, isoelectric focusing (IEF) and sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) are used in the first and second-dimensions, respectively (1). Traditionally, the first dimension, IEF, has been run by casting the gels in glass capillaries that are subsequently loaded into a tank vertically, with an upper and lower reservoir. Samples can be applied to the space above the gel, and separation occurs by applying an electric field across the two ends of the gel. After the required separation time, the gels are removed by extruding them from the capillaries using a water-filled syringe to displace them directly into a suitable equilibration buffer.