**2.9 Digestion**

332 Gel Electrophoresis – Advanced Techniques

was diluted to a volume of 100 µl and applied to the column. After 5 min incubation, the depleted sample was collected in an eppendorf tube by centrifugation at 784g. The total protein concentration before/after depletion was determined with Bio-Rad protein assay. After depletion of high abundant proteins the samples were desalted using PD-10 columns.

2-DE was performed using IPGphor and Multiphor (GE healthcare). Briefly, the proteins were resuspended in 150 μL of a 2-DE sample buffer containing 9 M urea, 65 mM DTT, 2% Pharmalyte (GE Healthcare), 4% CHAPS, and 1% bromophenol blue and then centrifuged at 4ºC and 23000g for 30 min to remove debris. The supernatant was then mixed with a rehydration buffer consisting of 8 M urea, 4% CHAPS, 0.5% IPG buffer 3-10 NL (GE Healthcare), 19 mM DTT, and 5.5 mM Orange G to a final volume of 350 μL. The first dimension was performed by in-gel rehydration for 12 h in 30 V on 18 cm pH 4-7 linear or pH 3-10 nonlinear IPG strips (Immobiline DryStrips, GE Healthcare). The proteins were then focused at 53000 Vh at a maximum voltage of 8000 V (Görg et al., 2000). The second dimension (SDS-PAGE) was performed by transferring the pI focused proteins (IPG strips) to homogeneous or gradient home-cast gels on gel bonds. The electrophoresis was

Sypro Ruby (Bio-Rad) staining were done according to manufacturer's instructions. In short, gels to be stained with Sypro Ruby were directly placed in a fixing solution containing 10% methanol and 7% acetic acid for at least 20 minutes after 2-DE. Gels were then washed 3x10 minutes under agitation with Milli-Q water before approximately 400 mL of Sypro Ruby

Silver staining of gels were done according to Shevchenko et al. 1996, with some few modifications. Proteins were fixed by incubating the gel in 50% methanol and 5% acetic acid for at least 20 minutes directly after 2-DE and then incubated with 50 % Methanol for 5 minutes, followed by Milli-Q water for 10 minutes. In the sensitizing step the gel was incubated with 0.02 % sodium thiosulphate for 1 minute, followed by 2x1 minutes washing with Milli-Q water. The gel was then immersed in 0.1 % silver nitrate solution for 20 minutes before excess of silver was washed away by 2x1 minute in Milli-Q water. Next, the gel was developed in 0.04 % formaldehyde in 2 % sodium bicarbonate solution for 2x1 minute. The exact developing time was optimized depending of the protein amount in the gel. Finally, the reaction was stopped by incubation 1x5 min in 0.5 % glycine and the gel

The images of the protein patterns were analyzed by a CCD (Charge-Coupled Device) camera digitizing at 1340\*1040 pixel resolution in a UV scanning illumination mode for Sypro Ruby stained gels or at 1024\*1024 pixel resolution in white light mode for silver stained gels using a Flour-S-Multi Imager in combination with a computerized imaging 12 bit system (PDQuest 2-D gel analysis software, version 7.1.1). The unit of the UV light source is expressed in counts while the unit of the white light source is expressed as optical density (OD). Gel images were evaluated by spot detection, spot intensities and geometric

**2.6 2-DE analysis**

performed at 40-800 V, 10°C, 20-40 mA, overnight.

stain were added and incubated in room temperature over night.

**2.7 Staining and image analysis** 

washed with Milli-Q water for 2x20 min.

properties.
