Protean 2 system

Bromophenol loading buffer was added to the PCR products and DGGE performed with a 20–80% denaturing gradient in 8% acrylamide gels (where 100% denaturant is 7 M urea and 40% formamide) in a Protean II system (Bio-Rad, USA) at 60°C, 100 V for 17 h. Gels were stained with ethidium bromide and the banding patterns recorded (ImaGo compact imaging system, B&L Systems, The Netherlands). The software package Phoretics 1D (version 2003.02; Nonlinear Dynamics) was used to analyze the banding patterns by removing background intensity from each lane, assigning standardized retardation factors (Rf) and determining the relative intensity of each band to the total intensity in each lane. DGGE bands selected for sequencing were excised and reamplified until single DGGE bands were obtained. Bands that could not be reamplified were manually excluded from analysis (e.g. heteroduplexes). Purification and sequencing (forward and reverse) of the PCR products was performed by Macrogen (Korea).

Proteins were separated by SDS-PAGE (8% polyacrylamide gels; Bio-rad, Protean II system), fixed in 50% ethanol, 12% acetic acid and stained in 0.08% Coomassie Brilliant Blue G-250, 20% ethanol, 8% ammonium sulfate, 0.8% phosphoric acid. Gels were de-stained using 20% ethanol and washed in water. Protein bands of interest were excised, fragmented with trypsin and peptide mixtures were electro-sprayed into an electrospray ion trap mass spectrometer (LCQ, Finnigan, San-Jose, CA). Mass spectrometry analysis was performed in Prof. Admon’s laboratory, Technion-Israel Institute of Technology, Haifa, Israel [56 (link)].

2-DE was carried out using the Multiphor system (Amersham Pharmacia) for IEF and Protean II system (Bio-Rad) for SDS-PAGE. Protein samples (150–200 μg) in 250 μl of solubilization solution (9 M urea, 2% CHAPS, 4 M thiourea, 2% IPG buffer; pH 4–7, 18 mM DDT and bromophenol blue) were loaded onto Immobiline Drystrips (13 cm, pH 4–7) and rehydration was preceded for 12 h at room temperature. IEF was conducted in gradient mode for 1 h at 1000 V, 1 h at 2000 V and 10 h at 8000 V, followed by 8000 V for a total of 65 kVh at 20°C. After the first-dimensional separation, the gel strips were equilibrated for 15 min in equilibration buffer (50 mM Tris-HCl pH 6.8, 6 M urea, 30% glycerol, 2% SDS and bromophenol blue). For the first equilibration, 0.25% DTT was added, and for the second equilibration, 4.5% iodoacetamide was used. SDS-PAGE was carried out in 12% separation gels with constant current of 40 mA/gel. After electrophoresis, the proteins were visualized by silver staining (GE Healthcare) and then the 2-DE images were obtained and analyzed using the Progenesis SameSpots program, v2.0 (Nonlinear Dynamics).

One-dimensional SDS-PAGE was carried out in this investigation to separate proteins based on size. Approximately 20 µg of extracted proteins was resolved using NuPAGE 4%–12% Bis-Tris Protein Gels (Invitrogen) and separated by SDS-PAGE according to Laemmli (1970) (link) on a Bio-Rad Protean II system. Loaded samples were run for 15 min at 100 V and later for 70 min at 150 V. Subsequently, gels were stained with Coomassie Stain G-250 and destained overnight for further protein pattern analysis according to band intensity.

For the protein spots identification 2-DE gels were used. Plasma samples were diluted in rehydration buffer and applied to pH 4–7 IPG strips, which were subsequently equilibrated as previously described [32 (link), 38 (link), 39 (link)]. For the second-dimension separation, 10% SDS-PAGE was performed according to Laemmli [40 (link)] using a Protean II system (BioRad) at 4°C and 25 mA/gel. The gels were fixed overnight and Silver Stained (GE Healthcare) following the manufacturer’s instructions and finally scanned with a GS-800 Calibrated Densitometer (BioRad).

The volume of the proteins extracts was adjusted to a final protein concentration of 500 μg in 340 μl of rehydration buffer. Subsequently, 1% DTT and 2% immobilized pH gradient (IPG) buffer pH 3–10 (Amersham Biosciences, Freiburg, Germany) were added. The protein sample was loaded onto 17- cm, non- linear pH 3–10 IEF isoelectric focusing strips (Immobiline DryStrip, Amersham Biosciences). IEF was performed using a IPGphor™ system (Amersham Biosciences) with the following program: 10 h at 20°C, 12 h at 30 V, 1 h at 500 V, 8 h at 1,000 V and 10 h at 8,000 V.
The strips were equilibrated for 15 min in 10 ml of equilibration solution (0.375 M Tris–HCl, pH 8.8, 6 M urea, 20% [v/v] glycerol and 2% [w/v] SDS), with 2% (w/v) DTT and for 15 min in 10 ml of equilibration solution containing 2% (w/v) iodoacetamide. Subsequently, the strip was transferred onto a 10% SDS-PAGE gel for second-dimension electrophoresis using a Bio-Rad Protean II system with 30 W per gel. The gels were subsequently stained with Coomassie brilliant blue.