Mechanical precision in two-dimensional electrophoresis can improve protein spot positional reproducibility.

Abstract

Current methods for high resolution two-dimensional electrophoresis (2DE) of proteins are capable of separating over 5000 protein spots in one procedure. Running and analysing such 2DE gels requires skilled technical work. However, the variable reproducibility of spot positions means that, even under the best circumstances, one gel cannot be overlain directly on another for precise comparison. Therefore, new and improved technologies that enhance gel-to-gel reproducibility are required. To this end, we have designed and built a research instrument to test whether a precise mechanical device could improve the gel-to-gel reproducibility by reducing the amount of distortion and positional variation between the first and second dimension gels. Other causes of poor reproducibility, including sample type and preparation, gel matrices and running conditions were not varied in order to limit this study to the mechanical variations inherent in current 2DE systems. We found that the sample standard deviation of pooled data for measured protein spot-to-spot distances in the prototype device was 1.3 mm as compared to 4.3 mm in a conventional 2DE system. These improvements support the possibility that greater automation of the multistep 2DE process will enhance reproducibility. This approach seems justified in order to achieve significantly better matching between gels and between results from different laboratories.