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2-DE is still the method of choice if complex protein mixtures are to be separated with high resolution, permitting the simultaneous analysis of hundreds or even thousands of gene products. The separation is based on the combination of two orthogonal separation principles. In the first dimension, proteins are separated due to their different isoelectric points and in the second dimension due to their molecular weight (Figure 12). 2-DE was performed using immobilized pH gradients (IPG-Dalt) based on the protocol of (Görg et al., 1988). In the first dimension, denaturated proteins were separated by isoelectric focusing (IEF) according to their intrinsic charge at a certain pH. Proteins have electrophoretic mobility as long as they reach the pH at which their intrinsic net charge becomes zero (isoelectric point, pI). Consequently they migrate in a stable pH gradient upon high voltage to the pH where they reach their isoelectric point. The pI of a specific protein depends on its content of basic and acidic amino acids. Commercially available IPG strips contain a pre-formed pH gradient immobilized in a homogeneous polyacrylamide gel.

In the second dimension the intrinsic charge of the proteins is shielded by the anionic detergent SDS and therefore separation of the proteins occurs due to their apparent molecular mass by gradient SDS-PAGE. Separated proteins can be visualized as protein spots by staining of the gel (see chapter 2.1.6).

2.1.5.1 Sample preparation

Pre-treatment of samples for 2-DE involves solubilization, denaturation and reduction to completely break up the interactions between the proteins (Rabilloud et al., 1997). Solubilization and denaturation of proteins was accomplished using 2-DE lysis buffer. It contains high molar concentrations of urea (9 M) and thiourea (2 M), as well as the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propansulfonate (CHAPS; 4% (w/v)) and is supplemented with dithioerythritol (DTE; 65 mM). The 2-DE lysis buffer was stored at -80°C.

Soluble fractions of light- and dark-adapted ROS were immunoprecipitated with anti-Rac1 and anti-RhoA antibodies (see chapter 2.1.8.2) and proteins were eluted with 210 µL 2-DE lysis buffer in microspin columns (GE Healthcare). The eluate was supplemented with 2.2 µL Pharmalyte (carrier ampholytes pH 3-10 for IEF, 0.36 meq/mL, GE Healthcare) and 1.1 µL bromophenol blue (0.5% (w/v)) and centrifuged for 30 min at 22°C and 50000 g (Beckman Optima ultracentrifuge; TLA110 rotor).

Figure 12: Separation principle of 2-DE gel electrophoresis. 1. dimension

IEF: separation of the proteins due to their different isoelectric points. 2. dimension SDS-PAGE: separation of proteins according to their molecular weight.

2.1.5.2 Rehydration and sample loading

7 cm IPG strips with a non-linear pH gradient covering pH values from 3-11 with a flat pH gradient at neutral pH values were used (GE Healthcare) for IEF. The sample (prepared as described in chapter 2.1.5.1) was pipetted air bubble free into the rehydration tray and the IPG strip was placed, with the gel matrix side facing down, directly into the sample solution. IPG strips were completely overlaid with gas-free mineral oil (DryStrip Cover Fluid, GE Healthcare), in order to exclude drying and formation of urea crystals.

2.1.5.3 Isoelectric focusing (IEF)

IEF was performed using the Multiphor II apparatus (GE Healthcare) with a water cooling system. For uniform,cooling, the Dry Strip Kit was laid air bubble free onto the cooling plate with 10 mL mineral oil to ensure equal contact. The focusing strip aligner was placed air bubble free into the Dry Strip Kit with 8 mL mineral oil in between. Surplus mineral oil, urea crystals and proteins that had not entered the gel matrix during the in-gel rehydration step were carefully removed from the IPG strips by dabbing with wet (ddH2O) filter papers

(Whatman). IPG strips were then laid gel-side-up into the grooves of the aligner. Wet (ddH2O) electrode strips (GE Healthcare) were placed at the anodic and cathodic ends of the

strips, respectively and electrodes were positioned upon the electrode strips. Subsequently, the IPG strips were overlaid with mineral oil. Isoelectric focusing was performed under cooling at 20 to 23°C. In the beginning, a low voltage step (150 V) was applied, in order to remove remaining salts from the strips. At higher voltages (up to 3500 V) proteins began to focus in the stable pH gradient. Focusing of the 7 cm stripes was continued until 10-15 kVh were reached (IEF-program: 30 V: slope/1 min, hold/16 h; 200 V: slope/1 min, hold/2 h; 3500 V: slope/2 h, hold/3 h). At this time point the steady state phase was reached, i.e. the time point

at which all the proteins were focused. After isoelectric focusing, IPG strips were used immediately for the second dimension or were frozen at -20°C between two sheets of plastic film until further use.

2.1.5.4 Equilibration and transfer of the IPG strips

After IEF, the IPG strips were rinsed with ddH2O and incubated in two steps in 2-DE

equilibration solutions to prepare the stripes for the second dimension (SDS-PAGE).In order to prevent electroendosmotic effects during the protein transfer from the focusing strip into the SDS gel, 2-DE equilibration solutions contained urea and glycerol (50 mM Tris-HCl pH 6.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS). In the first step, IPG strips were incubated

for 10 min at RT in reducing 2-DE equilibration solution containing 65 mM DTE (freshly prepared) resulting in cleavage of disulfide bonds between the sulfhydryl groups of the polypeptides. Subsequently the stripes were incubated for 10 min at RT in 2-DE equilibration solution supplemented with 260 mM 2-iodoacetamide and bromophenol blue. The resulting alkylation of free sulfhydryl groups, from excessive DTE and cystein residues, prevented the reformation of disulfide bonds. Following equilibration of the stripes in SDS electrophoresis buffer, they were transferred air bubble free on top of the stacking gel of a SDS-gradient gel (9-15%), with the gel side facing the smaller glass plate and with the acidic end of the strips oriented to the left side of the gel. For protein size determination a small piece of Whatman paper, soaked with 7 µL of pre-stained protein ladder (PageRuler Prestained Protein Ladder, Fermentas), was carefully positioned next to the strip.

Excessive SDS electrophoresis buffer was gently removed with Whatman paper and IPG strips were embedded air bubble free in agarose solution (0.5% (w/v) agarose in SDS electrophoresis buffer).

2.1.5.5 Second dimension: SDS-PAGE

2DE Electrophoresis was performed in a mini Protean III (BioRad) electrophoresis chamber with a voltage of 60 V until the proteins reached the separating gel. The voltage was elevated to 110 V until the bromophenol blue front reached the end of the gels (about 1,5 h). Following electrophoresis gels were stained using the silver staining method (see chapter 2.1.6.1)