Silver staining artifacts, unrelated to protein, such as horizontal lines and vertical micro or point streaking on two‐dimensional gels with carrier ampholytes or immobilized pH gradients in the first dimension are efficiently eliminated by adding iodoacetamide to the equilibration buffer of the first‐dimensional gel.

Leaf proteins from 14 barley cultivars (Hordeum vulgare) were analyzed by two-dimensional electrophoresis with immobilized pH gradients (IPG 4-7 and IPG 6-10) in the first dimension. Highly reproducible two-dimensional patterns were obtained, owing to constant spot positions along the isoelectric focusing axis. A number of variety-specific protein spots were detected, allowing us to discriminate barley cultivars not only into main groups but into individual cultivars.

Horizontal two-dimensional (2-D) electrophoresis with immobilized pH gradients (IPG) in the first dimension for buffer soluble proteins and for complex proteins solubilized in the presence of Nonidet P-40 (Görg et al., Electrophoresis 1987, 8, 45-51), has been extended to analyze basic proteins of yeast cells focused under non-equilibrium and equilibrium conditions. Transient state isoelectric focusing (IEF) in IPG gels revealed sample smearing and background staining, displaying horizontal streaks in the resultant 2-D patterns. Inclusion of 0.5% carrier ampholytes (CA) to the IPG gel (IPG-CA), resulted in the formation of many sharp protein bands after transient state IEF with resultant distinct spots in the 2-D patterns; however, resolution was poor and the gel contained heavy background staining. With prolonged focusing time, background staining disappeared and there was less difference in the final steady state IEF patterns obtained with IPG and IPG-CA. Reduction of the Immobiline concentration to one third the manufacturer's recommended amount did not improve IEF resolution with respect to streaking and background staining under either transient state or equilibrium conditions. In general, spot intensities were less on 2-D gels using diluted IPG gels than with "standard" IPG gels. Optimization of 2-D electrophoresis with IPGs in the first dimension was strongly related to IEF conditions. The use of IPG gels focused to equilibrium should not only improve inter-gel reproducibility and resolution but also the quality of the final 2-D patterns with respect to background staining and horizontal streaking.

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.

Horizontal two‐dimensional (2‐D) electrophoresis with immobilized pH gradients (IPG) in the first dimension, described for soluble proteins in Electrophoresis 1985, 6, 599–604, has been extended to an analysis of complex protein mixtures, such as leukemia cell proteins or bean proteins, in the presence of nonionic detergents. By optimizing the ratio of gel volumes for the first and second dimensional separation, and by decreasing the concentration of Nonidet P‐40 in the IPG gel, undistorted protein patterns in the 2‐D gel are obtained. Horizontal and vertical streaking are considerably diminished by optimized reswelling conditions of the dry IPG strips in presence of both urea and detergent. Resolution is improved and a reduced background is obtained when overcrowded gel areas are spread by flattening the pH gradient (in the first dimension) and the polyacrylamide gradient (in the second dimension).