Solubilization of proteins from human lymph node tissue and two-dimensional gel storage

Autor(es)

Marqui, Alessandra Bernadete Trovó de ; Vidotto, Alessandra ; Polachini, Giovana Mussi ; Bellato, Cláudia de Mattos ; Cabral, Hamilton ; Leopoldino, Andréia Machado ; Góis Filho, José Francisco de ; Fukuyama, Érica Erina ; Settanni, Flávio Aurélio Parente ; et al.

• Tipo de documento: Comunicação Científica
• Fonte: Journal of Biochemistry and Molecular Biology; v. 39, n. 2, p. 216-222, Mar. 2006.
• Área do conhecimento: Ciências Biológicas - Genética
• Assunto(s): Genética médica Sistema linfático Linfonodos

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) 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 (Ndecyl-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. (AU)

• Processo FAPESP: 01/14473-0 - Analise proteomica em neurofibromatose tipo 1.
• Beneficiário: Eloiza Helena Tajara da Silva
• Modalidade de apoio: Auxílio à Pesquisa - Regular