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.
Corbett
J M
JM
National Heart and Lung Institute, Heart Science Centre, Harefield Hospital, Middlesex, UK.
Dunn
M J
MJ
Posch
A
A
Görg
A
A
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods / Q000592:standards*; D006352:Heart Ventricles; D001467:Hordeum / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007525:Isoelectric Focusing / Q000379:methods / Q000592:standards*; D007753:Laboratories / Q000592:standards*; D009124:Muscle Proteins / Q000032:analysis / Q000302:isolation & purification; D009206:Myocardium / Q000737:chemistry; D018515:Plant Leaves; D010940:Plant Proteins / Q000032:analysis / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012984:Software; D013194:Staining and Labeling
D007202:Indicators and Reagents; D009124:Muscle Proteins; D010940:Plant Proteins; D011506:Proteins