Towards validating a method for two-dimensional electrophoresis/silver staining

Two-dimensional electrophoresis (2-DE) is a technique involving numerous steps, many of them to be performed manually. Hence, some operator dependency must be taken into account. An attempt to elucidate the reliability of 2-DE combined with silver staining is presented, employing the general practice to validate a method in pharmaceutical analysis. Most proteomic studies employing 2-DE aim at qualitative or quantitative differences in protein expression. One of the most sensitive and broadly applied staining techniques is silver staining. In order to gain information on accuracy, precision, linearity, and ruggedness of this technique, gels were run in replicates with different amounts of protein from a complex standard sample. In addition, sets of gels were repeated by two different operators in a second independent laboratory equipped with identical hardware and software. Our results show that reliable qualitative data on differential protein expression can be obtained by 2-DE, nevertheless replicate gels should be run and experimental conditions have to be kept stringently to a standardized protocol. Quantitative data are just achievable with spots, which are well-resolved, of high quality, with an optical density (OD) above a certain threshold (OD > 10), and which show a linear response. Quantitative differences occurring due to method-derived deviations may easily be misinterpreted as true changes in protein expression. After normalization, relative standard deviation (RSD) values of approximately 30% (n = 4) could be obtained, therefore minor changes (< 50%) should be critically reviewed.