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

Publisher Website
Google Scholar
Abstract
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 ∼ 1.5 mm). Nevertheless, IPG‐DALT provides significantly improved reproducibility of spot positions compared to conventional isoelectric focusing with synthetic carrier ampholytes.