Proteomic approaches to identify cold-regulated soluble proteins

Abstract

DIGE (differential in-gel electrophoresis) is a modifi ed version of the widely used 2-D gel electrophoresis (2-DE) for separation of complex protein samples. Two extracts to be compared are differentially labeled using fl uorescent cyanine dyes and then separated together by 2-DE. An internal standard labeled using a third dye is included. This approach avoids the pitfalls of gel distortions frequently observed in the standard procedure, which hamper the subsequent gel image analysis. Inclusion of an internal standard improves the quantitative evaluation of the protein patterns. Using the advantages of the DIGE approach, impact of minor temperature differences during cold stress treatment could be quantitatively monitored. We will describe the application of DIGE to monitor the impact of cold stress on the proteome pattern of Arabidopsis. In addition to the separation of proteins, we will also outline how plant growth is performed. Finally, we will also give protocols how proteins of interest can be identifi ed by MALDI-TOF- as well as ESI-MS/MS.