Proteomic approaches to identify cold-regulated soluble proteins.
DIGE (differential in-gel electrophoresis) is a modified 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 fluorescent 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 identified by MALDI-TOF- as well as ESI-MS/MS.
Department of Physiology and Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstraße 3, 06466, Stadt Seeland, OT Gatersleben, Germany.
Stefanie
S
Döll
Rico
R
Lippmann
Hans-Peter
HP
Mock
D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002232:Carbocyanines / Q000737:chemistry; D002853:Chromatography, Liquid; D003080:Cold Temperature; D004396:Coloring Agents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D029681:Arabidopsis Proteins; D002232:Carbocyanines; D004396:Coloring Agents; D020543:Proteome; D014357:Trypsin