| Full text | |
| Author(s): |
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.
Total Authors: 10
|
| Document type: | Research communication |
| Source: | Journal of Biochemistry and Molecular Biology; v. 39, n. 2, p. 216-222, Mar. 2006. |
| Field of knowledge: | Biological Sciences - Genetics |
| Abstract | |
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) | |