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Solubilization of Proteins from Human Lymph Node Tissue and Two-Dimensional Gel Storage
De Marqui, Alessandra Bernadete Trovo,Vidotto, Alessandra,Polachini, Giovana Mussi,De Mattos Bellato, Claudia,Cabral, Hamilton,Leopoldino, Andreia Machado,De Gois Filho, Jose Francisco,Fukuyama, Erica Korean Society for Biochemistry and Molecular Biol 2006 Journal of biochemistry and molecular biology Vol.39 No.2
In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT(dithiothreitol) and 0.2% carrier ampholytes; (b) 5M urea, 2M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), 40mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7M urea, 2M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.
Solubilization of Proteins from Human Lymph Node Tissue and Two-Dimensional Gel Storage
Alessandra Bernadete Trovo De Marqui,Alessandra Vidotto,Giovana Mussi Polachini,Claudia De Mattos Bellato,Hamilton Cabral,Andreia Machado Leopoldino,Jose Francisco De Gois Filho,Erica Erina Fukuyama,F 한국생활환경학회 2006 BMB Reports Vol.39 No.2