POSITIONAL REPRODUCIBILITY OF PROTEIN SPOTS IN 2-DIMENSIONAL POLYACRYLAMIDE-GEL ELECTROPHORESIS USING IMMOBILIZED 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. Aliquo ts of two different samples, human cardiac and barley leaf proteins, w ere separated in two different laboratories (London and Munich), using 180 mm long IPG gel strips, pH 4-8, for the first dimension and homog eneous 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 posi tional reproducibility. The IPG-dimension was highly reproducible in e ach laboratory, with a mean standard deviation of about 1 mm for both types of sample. Interlaboratory comparisons revealed identical result s for barley with a mean standard deviation along the x-axis of about 1 mm, whereas the cardiac matchset showed slightly more variability (m ean standard deviation similar to 1.5 mm). Nevertheless, IPG-DALT prov ides significantly improved reproducibility of spot positions compared to conventional isoelectric focusing with synthetic carrier ampholyte s.