Reduction of proteins during sample preparation and two-dimensional gel electrophoresis of woody plant samples.
Protein extraction procedure and the reducing agent content (DTT, dithioerythritol, tributyl phosphine and tris (2-carboxyethyl) phosphine (TCEP)) of the sample and rehydration buffers were optimised for European beech leaves and roots and Norway spruce needles. Optimal extraction was achieved with 100 mM DTT for leaves and needles and a mixture of 2 mM TCEP and 50 mM DTT for roots. Performing IEF in buffers containing hydroxyethyldisulphide significantly enhanced the quality of separation for all proteins except for acidic root proteins, which were optimally focused in the same buffer as extracted.
Vâlcu
Cristina-Maria
CM
Section of Forest Genetics, Technische Universität München, Freising-Weihenstephan, Germany. valcu@wzw.tum.de
Schlink
Katja
K
D002021:Buffers; D004229:Dithiothreitol / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D029964:Fagus / Q000737:chemistry*; D007202:Indicators and Reagents / Q000737:chemistry; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry; D028222:Picea / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D018514:Plant Structures / Q000737:chemistry
D002021:Buffers; D004229:Dithiothreitol; D007202:Indicators and Reagents; D010720:Phosphines; D010940:Plant Proteins; C043255:tri-n-butylphosphine; C080938:tris(2-carboxyethyl)phosphine