Comparative Two-Dimensional Fluorescence Gel Electrophoresis.

Abstract

Two-dimensional comparative fluorescence gel electrophoresis (CoFGE) uses an internal standard to increase the reproducibility of coordinate assignment for protein spots visualized on 2D polyacrylamide gels. This is particularly important for samples that need to be compared without the availability of replicates and thus cannot be studied using differential gel electrophoresis (DIGE). CoFGE corrects for gel-to-gel variability by co-running with the sample proteome a standardized marker grid of 80-100 nodes, which is formed by a set of purified proteins. Differentiating of reference and analyte is possible by the use of two fluorescent dyes. Variations in the y-dimension (molecular weight) are corrected by the marker grid. For the optional control of the x-dimension (pI), azo-dyes can be used. Experiments are possible in both vertical and horizontal (h) electrophoresis devices, but hCoFGE is much easier to perform. The CoFGE experimental principle can additionally be used for protein quantification. For data analysis, commercial software has been adapted.