METHODS
Two-dimensional gel electrophoresis (2-DE)
The protocol used for proteomics analysis is similar as that described before¹ ². Cells were scraped and centrifuged at 4C, 13.2krpm, for 1 minute. Cell pellets were lysed in 2D lysis buffer (9.5 M urea, 2% w/v CHAPS. 0.8% w/v Pharmalyte, pH 3-10 and 1% w/v DTT) for 30 minutes, and centrifuged for 20 minutes at 20C, 13.2krpm. Supernatant was divided into aliquots and protein concentration was determined. Extracts were diluted directly with rehydration solution (8 M urea, 0.5% w/v CHAPS, 0.2% w/v DTT, and 0.2 % w/v Pharmalyte pH 3-10) or subject to an additional clean-up procedure by using 2D PrepReady CleanUp Kit (Bio-Rad) to remove contaminants interfering with IEF.
Protein samples were loaded on 18cm nonlinear immobilized pH gradient strips, pH 3-10 (Immobiline DryStrips, GE healthcare), 100μg total protein for analytical gel and 400μg for preparative gel. After overnight rehydration, strips were focused in Multiphor™ II IEF System (GE healthcare) at 0.05 mA / IPG strip for 60 kVhrs at 20C.
Once IEF was finished, the strips were equilibrated in equilibration buffer (6M urea, 30% v/v glycerol, 2% w/v SDS and 0.01% w/v Bromphenol blue) with addition of 1% (w/v) DTT for 15 min, followed by a further 15 min equilibration in the same buffer containing 4.8% (w/v) iodoacetamide in place of DTT.
SDS-PAGE was performed using 12%T, 2.6% C polyacrylamide gels without a stacking gel and the Ettan™ DALTsix vertical electrophoresis system (GE healthcare). The second dimension was carried out at 10C until the Bromphenol blue dye front had migrated off the lower end of the gels.
Gels were stained by silver staining (PlusOne™ Silver Staining Kit, Protein, GE healthcare) using a slightly modified protocol³, which is compatible with mass spectrometry.
[1] Dunn, M. J., Methods Mol Biol 1997, 64, 25-36.
[2] McGregor, E., Kempster, L., Wait, R., Welson, S. Y. et al., Proteomics 2001, 1, 1405-1414.
[3] Yan, J. X., Wait, R., Berkelman, T., Harry, R. A. et al., Electrophoresis 2000, 21, 3666-3672.