title: A competent extraction method of plant proteins for 2-D gel electrophoresis.
abstract: The efficient extraction of high-quality proteins is a key factor for a successful proteomic analysis approach. In the method suggested here, absolute ethanol containing 10 mM DTT was used to precipitate the proteins in plant tissue homogenates followed by their resuspension in a urea-/thiourea- and NP-40-containing solution. Protein profiles were examined on pH 3-11 non-linear IEF strips and SDS-PAGE and compared with extracts using the established method of acetone-10% TCA/0.07% 2-mercaptoethanol precipitation (V. Méchin et al., Methods Mol. Biol. 2006, 355, 1-8). In addition to protein profile similarity for the two extracts, the acidic part of the acetone containing 10% TCA/0.07% 2-mercaptoethanol extraction showed protein spots with high molecular weight in the range of 250-150 kDa, while the ethanol containing 10 mM DTT extracts indicated extra proteins spots at the basic part of the gels with molecular weights in the range of 25-15 kDa. The MALDI-TOF-MS of differential spots from acetone containing 10% TCA/0.07% 2-mercaptoethanol precipitation method and absolute ethanol containing 10mM DTT indicated no similarity, ruling out the possibility that the two clusters shown represent identical proteins. The described method is easy in implementation, chemicals used are less toxic and proteins are easier to resuspend therefore presents an additional choice to implement towards finding the optimum method for extraction.
authors: Biodiversity and Climate Research Centre (BiK-F), Frankfurt am Main, Germany. ashoub@em.uni-frankfurt.de
authors: Ahmed
authors: A
authors: Ashoub
authors: Thomas
authors: T
authors: Berberich
authors: Tobias
authors: T
authors: Beckhaus
authors: Wolfgang
authors: W
authors: Brüggemann
mesh_terms: D000096:Acetone / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D000431:Ethanol / Q000737:chemistry; D001467:Hordeum / Q000737:chemistry; D008623:Mercaptoethanol / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D014238:Trichloroacetic Acid / Q000737:chemistry
chemical_list: D010940:Plant Proteins; D000096:Acetone; D000431:Ethanol; D014238:Trichloroacetic Acid; D008623:Mercaptoethanol