Positional reproducibility of protein spots in two-dimensional polyacrylamide gel electrophoresis using immobilised pH gradient isoelectric focusing in the first dimension: an interlaboratory comparison.

An intra- and interlaboratory comparison of positional reproducibility of protein spots in two-dimensional electrophoresis using immobilised pH gradients (IPG) in the first dimension (IPG-DALT) was made. Aliquots of two different samples, human cardiac and barley leaf proteins, were separated in two different laboratories (London and Munich), using 180 mm long IPG gel strips, pH 4-8, for the first dimension and homogeneous SDS-PAGE gels (12% T) for the second dimension. Subsets of 340 (cardiac) and 200 (barley) well-resolved spots distributed across the 2-D gel patterns were selected for computer analysis (PDQUEST) of positional reproducibility. The IPG-dimension was highly reproducible in each laboratory, with a mean standard deviation of about 1 mm for both types of sample. Interlaboratory comparisons revealed identical results for barley with a mean standard deviation along the x-axis of about 1 mm, whereas the cardiac matchset showed slightly more variability (mean standard deviation approximately 1.5 mm). Nevertheless, IPG-DALT provides significantly improved reproducibility of spot positions compared to conventional isoelectric focusing with synthetic carrier ampholytes.