Comparison Of Three Nuclear Protein Extraction Methods Used In2-dimensional Electrophoresis

BackgroundWith the development of proteomics, the studies of proteomics have reached the subcellular level. However, the resolution of the current technology for whole cell proteome analysis could not fulfill such requirements; as a result, subcellular proteomics is growing as the new trend for proteomics. Subcellular proteomics can not only reduce the complexity of the proteins, but also can enrich the low-abundance proteins, and improve the detection rate of low-abundance proteins, which will help better understand the mechanisms of cell physiology. Nuclear proteomics is part of the subcellular proteomics, and the acquirement of pure nuclear proteins is crucial for such study.ObjectiveCompare the efficiency of three different eukaryotic nuclear protein extraction methods and their applicability for two-dimensional electrophoresis (2-DE).Methods1. Nuclear proteins of HeLa cells were extracted by the Dounce homogenizer, commercial extract kit, and votexing method, respectively.2. P rotein concentration was determined by the Bradford method. Representative nuclear and cytoplasmic proteins were detected by Western blot.3. The extracted nuclear proteins were subjected to2-DE, and the obtained gel images were analyzed by PDQuest8.0.ResultsVotexing method used lest time to extract proteins. The Dounce homogenizer method obtained less nuclear proteins with more cytoplasmic protein contamination than the other two methods.2-DE protein profiles were similar for the nuclear proteins extracted by the three different methods.ConclusionThe three nuclear protein extract methods can be used for2-DE in proteomic analysis. However, methods using commercial kit and votexing are more efficient, and the votexing method is also cost-efficient.