Difference Gel Electrophoresis ( DIGE )

Abstract One of the largest challenges in proteomics today is to be able to quantify the composition and amount of proteins found in a specific cell or tissue at a defined time point. Difference gel electrophoresis (DIGE) is a gel electrophoresis‐based technique for protein quantification in complex mixtures. In DIGE the high resolution of two‐dimensional gel electrophoresis is combined with the excellent dynamic range obtained by fluorescent tag labelling of protein samples. The output of DIGE experiments provides information about how many proteins display changed expression levels on a specific treatment. In addition, proteins of interest can be excised and identified with conventional mass spectrometry techniques and further analysed by other biochemical methods. Key concepts: DIGE is a gel‐based technique for relative protein quantification in complex protein samples. Experimental design, sample preparation and data analysis are all critical steps of a DIGE experiment. Labelling of protein samples is performed by either a minimal or saturation labelling procedure. Use of an internal standard minimizes gel to gel variation and provides increased power to the experiment. For protein identification poststaining of gels is necessary when a minimal labelling procedure has been used. DIGE can also be used for other applications such as native gel electrophoresis of protein complexes. The DIGE technique is constantly being improved and continues to be an important method in functional proteomics.