Two-dimensional electrophresis is separating the same sample with two distinct separation techniques or two different electrophoresis separations. The separated bands from one electrophoresis are resolved more with the second electrophoresis.
IEF followed by PAGE or AGE is the most frequent two-dimensional electrophoresis. The gel from the IEF capillary is removed and placed across the PAGE or AGE gel slab at right angles for the second electrophoresis. If PAGE is used for the second electrophoresis, it is often PAGE with SDS.
Two-dimensional electrophoresis can also be a single sample run on either agarose or polyacrylamide gels. The gel is then turned 90 degrees and the same type electrophoresis is run on the separated solutes to separate each band from the first run into more bands.
The image below shows a two-dimensional electrophoresis separation of proteins which is IEF followed by PAGE with SDS. The proteins were first separated by IEF on a very narrow gel strip. This strip was then positioned at top of a polyacrylamide gel with SDS for the second electrophoresis. The IEF gel is the very narrow strip on top and remainder of the image is the many separated proteins on the PAGE with SDS.