Solubilization of proteins from human lymph node tissue and two-dimensional gel storage.
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Data
2006-03-31
Autores
de Marqui, Alessandra Bernadete Trovó
Vidotto, Alessandra
Polachini, Giovana Mussi
Bellato, Cláudia de Mattos
Cabral, Hamilton
Leopoldino, Andréia Machado
de Góis Filho, José Francisco
Fukuyama, Erica Erina
Settanni, Flávio Aurélio Parente
Cury, Patrícia Maluf
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Resumo
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) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M 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.
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buffer, detergent, protein, tumor protein, cell, chemistry, comparative study, head and neck tumor, human, isoelectric focusing, lymph node, methodology, solubility, squamous cell carcinoma, standard, time, two dimensional gel electrophoresis, Buffers, Carcinoma, Squamous Cell, Cells, Detergents, Electrophoresis, Gel, Two-Dimensional, Head and Neck Neoplasms, Humans, Isoelectric Focusing, Lymph Nodes, Neoplasm Proteins, Proteins, Solubility, Time
Como citar
Journal of biochemistry and molecular biology., v. 39, n. 2, p. 216-222, 2006.