Solubilization of proteins from human lymph node tissue and two-dimensional gel storage.
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.
Universidade Estadual Paulista, Instituto de Biociências, Letras e Ciências Exatas, Departamento de Biologia, São José do Rio Preto, SP, Brazil.
Alessandra Bernadete Trovó
AB
de Marqui
Alessandra
A
Vidotto
Giovana Mussi
GM
Polachini
Cláudia de Mattos
Cde M
Bellato
Hamilton
H
Cabral
Andréia Machado
AM
Leopoldino
José Francisco
JF
de Góis Filho
Erica Erina
EE
Fukuyama
Flávio Aurélio Parente
FA
Settanni
Patrícia Maluf
PM
Cury
Gustavo Orlando
GO
Bonilla-Rodriguez
Mario Sergio
MS
Palma
Eloiza Helena
EH
Tajara
D002021:Buffers; D002294:Carcinoma, Squamous Cell / Q000737:chemistry*; D002477:Cells; D003902:Detergents / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods* / Q000592:standards; D006258:Head and Neck Neoplasms / Q000737:chemistry*; D006801:Humans; D007525:Isoelectric Focusing; D008198:Lymph Nodes / Q000737:chemistry*; D009363:Neoplasm Proteins / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012995:Solubility; D013995:Time
D002021:Buffers; D003902:Detergents; D009363:Neoplasm Proteins; D011506:Proteins