Improved horizontal two‐dimensional electrophoresis with hybrid isoelectric focusing in immobilized pH gradients in the first dimension and laying‐on transfer to the second dimension

Two‐dimensional (2‐D) maps obtained with horizontal or vertical electrophoretic systems are compared. There are no differences in resolution, spot size and distribution, but significant differences in handling, Horizontal 2‐D electrophoresis, where the equilibrated isoelectric focusing (IEF) gel strip with plastic backing is applied to the surface of the horizontal sodium dodecyl sulfate (SDS)‐gel, is extremely simple and easy to perform. Due to the optimal flat‐to‐flat contact of the two gels, no size or edge trimming of the gels, no agarose overlays nor embedding gels are needed. The influence of IEF, performed with carrier ampholytes, Immobilines or immobilized pH gradients (IPG) with carrier ampholytes, on 2‐D maps is demonstrated. More protein has migrated into the IPG gel with carrier ampholytes, displaying an increased number of spots.