Comparative two-dimensional fluorescence gel electrophoresis
Abstract Comparative two-dimensional gel electrophoresis (CoFGE) is a special version of two-dimensional polyacrylamide GE (2D-PAGE) and related to difference GE (2D-DIGE). It provides reproducibility and standardisation for 2D-PAGE by introducing a reference to the experiment. CoFGE uses different fluorescent labels to distinguish analyte and a marker protein mixture. The method allows in silico correction of the assignment of gel-separated proteins based on the co-run references, which form a grid of landmarks across the entire gel. The variability of spot coordinates is reduced to ~1% error and data can thus be compared to results generated independently with the same method. In this way, searchable repositories of gel-separated proteins become feasible. In addition, the CoFGE experimental principle can be used for protein quantification by applying the proteins of the marker grid in different concentrations. Here we present the protocol for conducting a CoFGE experiment, which takes about 2 days to complete for a technician skilled in GE.