Isoelectric focusing management: an investigation for salt interference and an algorithm for optimization

Two-dimensional electrophoresis (2-DE) has evolved into a robust separation technique in proteomic research. However, one of the major challenges in 2-DE experiments, the reproducibility of the first dimensional electrophoresis (IEF), has remained unsolved. It is well-known that the quality of IEF experiments is significantly affected by the salt interference. Nevertheless, the interference mechanisms of salts in IEF have never been systematically investigated. In this study, we comprehensively investigated the interference effects in IEF due to various kinds of simple and buffer salts in protein samples. Two interference schemes were proposed accordingly to elucidate the interference mechanisms of salts in IEF. Furthermore, to increase the reproducibility of IEF, we proposed that conductivity measurement is a feasible method to assess the salt content of 2-DE samples and developed an algorithm to predict the optimal total volt-hours (Vh) required for protein focusing in IEF. The developed algorithm had been evaluated under various IEF conditions for a variety of 2-DE samples and proven to be a reliable guide. In sum, information disclosed in this study should be of use for increasing the reproducibility and thus the applicability of 2-DE in current proteomics.