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Sequential solubilization of proteins precipitated with trichloroacetic acid in acetone from cultured Catharanthus roseus cells yields 52% more spots after two‐dimensional electrophoresis
Author(s) -
Jacobs Denise I.,
Rijssen Margriet S. van,
Heijden Robert van der,
Verpoorte Robert
Publication year - 2001
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/1615-9861(200111)1:11<1345::aid-prot1345>3.0.co;2-f
Subject(s) - trichloroacetic acid , solubilization , chromatography , chemistry , spots , acetone , proteome , thiourea , protein precipitation , catharanthus roseus , urea , electrophoresis , precipitation , solubility , biochemistry , high performance liquid chromatography , organic chemistry , physics , meteorology
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.