Sequential solubilization of proteins precipitated with trichloroacetic acid in acetone from cultured Catharanthus roseus cells yields 52% more spots after two-dimensional electrophoresis.
Sample preparation is still the most critical step in two-dimensional gel electrophoresis (2-DE), and needs to be optimized for each type of sample. To analyze the proteome of the medicinal plant Catharanthus roseus, we developed and evaluated a sequential solubilization procedure for the solubilization of proteins after precipitation in trichloroacetic acid and acetone. The procedure includes solubilization with a conventional urea buffer followed by a stronger solubilizing buffer containing thiourea. The sequential solubilization of the precipitated proteins results in very different spot patterns following 2-DE. The number of protein spots which could be detected in both samples of the sequential solubilization was only about 10% of the total number of spots. Compared to solubilization in a single step, the total number of spots that could be detected in the sequential solubilization procedure was increased by 52%. The method described is simple and is applicable to different types of plant tissue.
Jacobs
D I
DI
Division of Pharmacognosy, Leiden/Amsterdam Center of Drug Research, The Netherlands. d.jacobs@lacdr.leidenuniv.nl
van Rijssen
M S
MS
van der Heijden
R
R
Verpoorte
R
R
D000096:Acetone / Q000494:pharmacology*; D000470:Alkaloids / Q000378:metabolism; D030008:Catharanthus / Q000166:cytology* / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D014238:Trichloroacetic Acid / Q000494:pharmacology*
D000470:Alkaloids; D000096:Acetone; D014238:Trichloroacetic Acid