Technical variability of 2D gel electrophoresis – Application to soybean allergens

• 2-DE is robust and suitable for comparing the GM soybean with its non-GM counterpart; its technical variability is lower than the biological variability.
• Main source of variability is the gels so 3–4 gel replicates should be used.
• Other sources of variability are minor, which gives some experimental flexibility, i.e. study can be run over several days, several operators.

Two-dimensional gel electrophoresis (2-DE) technique is used as a performing technique to assess the variability of protein expression in crops, and especially soybean endogenous food allergens, which are a subset of proteins of interest for assessing whether genetically modified (GM) soybean has a different allergenic profile compared to its non-GM counterpart.On top of the biological variability of the 2-DE, which has already been studied by several laboratories, technical variability has to be evaluated. In this study, several sources of variability (number of gel replicates, protein extracts, study timings and operators) were assessed qualitatively and quantitatively on all detectable polypeptide spots as well as on food allergen spots. Results showed that the major source of variability was the number of gel replicates. Other sources were minor.This has a direct practical impact on the laboratory work as this supports the utilization of three or four gel replicates to get robust results. Furthermore, this implies that the study can be run over several days, and be performed by several trained operators, without impacting its reproducibility.Furthermore, 2-DE could detect a 2-fold change between two samples with an acceptable rate of false positives (below 7%). This level of sensitivity is acceptable in the context of safety assessment of GM soybean as the biological variability of proteins in soybean is higher than the technical variability shown in this study.Overall, the 2-DE technique is suitable for investigating endogenous food allergen variability between several soybean seeds, including GM and non-GM counterpart.