The E. coli strain RB791 was grown in LB medium and cells (50-100 ml) collected at different growth stages. After a centrifugation (10 min, 8000 rpm, 4 °C) the cells were washed twice with 2 ml TE-PMSF (1 mM EDTA, 0.1 M Tris, 14 µM PMSF, pH 7.5). The pellet was then resuspended in 3 ml TE-PMSF, disrupted by French Press with 900 PSI (62.1 bar) and the cell lysate centrifuged (30 min, 15.300 rpm, 4 °C). The supernatant was collected and the protein concentration determined with a commercially available kit (RotiNanoquant) according to Bradford, 1976. Aliquots of 100 µg were dried (SpeedVac) and stored at - 20 °C.
Prior to separation in the first dimension (IEF) the proteins were resuspended in 350 µl urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10, 100 mM Tris/HCl, pH 7.5, 1 mM EDTA, 14 µM PMSF) and transfered by in-gel rehydration into the IEF gels.

For the analysis of the fermentative growth the S. cerevisiae wild type strain BJ1991 was cultivated on rich YPD (1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) D-glucose) and collected at an optical density (600 nm) of 1.1. For the study of the respiration a wild type culture was grown in YPD to an optical density (600 nm) of 1-2, then pelleted, and transferred to YPG containing 3% (v/v) glycerol instead of D-glucose. After 16 h cells were collected, centrifuged (10 min, 8000 rpm, 4 °C) and washed twice with 2 ml TE-PMSF (1 mM EDTA, 0.1 M Tris, 14 µM PMSF, pH 7.5). Then the pellet was resuspended in 3 ml TE-PMSF and disrupted by French Press with 900 PSI (62.1 bar). The cell lysate was centrifugated (30 min, 15.300 rpm, 4 °C) and the supernatant collected. The protein concentration was determined with a commercially available kit (RotiNanoquant) according to Bradford, 1976. Aliquots of 100 µg were dried (SpeedVac) and stored at - 20 °C.
Prior to separation in the first dimension (IEF) the proteins were resuspended in 350 µl urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10, 100 mM Tris/HCl, pH 7.5, 1 mM EDTA, 14 µM PMSF) and transfered by in-gel rehydration into the IEF gels.

Yeast cells were grown and collected as described above. The mitochondria were isolated according to Meisinger et al., 2000. In brief, cells were pelleted by centrifugation (3.000 g, 5 min), washed with distilled water and resuspended under slowly shaking for 20 min at 30 °C in 2ml/ g DTT buffer (100 mM Tris-H2SO4 pH 9.4, 10 mM DTT). After washing with zymolyase buffer (1.2 M sorbitol; 20 mM potassium phosphate, pH 7.4) cells were incubated with 5mg/ g (w/v) Zymolyase-20T (Seikagaku Kogyo Co.) in 7 ml/ g zymolyase buffer for 45 min at 30 °C for conversation into spheroblasts. Homogenization was carried out by 15 strokes in a glass-Teflon potter in 6.5 ml/ g ice-cold homogenisation buffer (0.6 M sorbitol; 10 mM Tris-HCl, pH 7.4; 1 mM EDTA, 1 mM PMSF; 0.2 % (w/v) bovine serum albumin). This homogenate was diluted with 1 vol ice-cold homogenisation buffer and cell debris and nuclei removed by centrifugation (1500 g, 5 min, 4 °C). The supernatant was again centrifuged (3000 g, 5 min, 4 °C) and the mitochondria in the supernatant pelleted by additional centrifugation (12.000 g, 15 min, 4 °C). The pellet was washed with SEM (250 mM sucrose; 1 mM EDTA; 10 mM MOPS, pH 7.2), again centrifuged (12.000 g, 15 min, 4 °C) and resuspended in SEM to give a final concentration of 5 mg/ ml. The mitochondrial fraction was treated by 10 strokes in a glass-Teflon potter and loaded onto a three-step sucrose gradient (1.5 ml 60 %, 4 ml 32 %, 1.5 ml 23 %, 1.5 ml 15 % (w/v) sucrose in EM buffer (10 mM MOPS, pH 7.2; 1 mM EDTA). After centrifugation (134.000 g, 1 h, 2 °C) the purified mitochondria were recovered from the 60 %/ 32 % interface, diluted with 2 vol SEM and centrifuged (10.000g, 2 °C). The mitochondrial pellet was resuspended in urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 14 µM PMSF) and the protein concentration determined. Protein aliquots (100 µg) were stored at -20 °C.
Prior to separation in the first dimension (IEF) the volume of the urea buffer was adjusted to 350 µl and the proteins transfered by in-gel rehydration into the IEF gels.

Kidneys of adult mice (14.5 days) were dissected, washed in 5 vol of ice-cold 20 mM Tris/HCl pH 7.8, 10 mM EDTA, 2 mM EGTA, 2 mM DTT, dried (SpeedVac) and resuspended in urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 14 µM PMSF). The protein concentration was determined and aliquots of 100 µg stored at - 20 °C.
Prior to separation in the first dimension (IEF) the volume of the urea buffer was adjusted to 350 µl and the proteins transfered by in-gel rehydration into the IEF gels.

Bones (calvaria and femur) from 3-4 week old mice were collected, freezed with liquid nitrogen and grinded with a pestle to a fine powder. Proteins were precipitated with acetone (80 % acetone, 0.12 % (w/v) DTT) for at least 2 h at – 20 °C, pelleted by centrifugation (30 min, 14.000 rpm, 4 °C) and resuspended in urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 14 µM PMSF). The protein concentration was determined and aliquots of 100 µg stored at - 20 °C.
Prior to separation in the first dimension (IEF) the IEF gels were incubated in 350 µl urea buffer without protein. Prior separation the protein solution was applied into sample cups placed at the anodic end of the IEF gel.

Spleen tissue was homogenized, lymphocytes (B- and T-cells) were isolated using a lymphoprep-gradient centrifugation. The purified lymphocytes were added to a T cell purification column and the T-cells collected and centrifuged. The pellet was resuspended in urea buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 1 % (w/v) DTT, 2 % (v/v) carrier ampholytes 3-10, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 14 µM PMSF) according to Görg et al., 2000. The protein concentration was determined and aliquots of 100 µg stored at - 20 °C.
For the separation in the first dimension (IEF) the IEF gels were incubated in 350 µl rehydration solution (6 M urea, 2 M thiourea, 1% (w/v) CHAPS, 0.4 % (w/v) DTT, 0.5 % (v/v) carrier ampholytes 3-10) according to Görg et al., 2000 without protein. Prior to separation in the first dimension (IEF) the protein solution was applied into sample cups placed at the anodic end of the IEF gel.

A culture of a mouse fibroblast derived cell line (NIH3T3) was grown for 48 h in Dulbecco`s modified Eagle medium (DMEM) containing 3 % fetal calf serum (FCS). Cells were rinsed once with DMEM without FCS and collected from the plates (10 cm) by carefully scratching. Proteins were precipitated with acetone (80 % acetone, 0.12 % (w/v) DTT) for at least 2 h at – 20 °C, pelleted by centrifugation (30 min, 14.000 rpm, 4 °C) and resuspended in urea buffer (8 M urea, 2 M thiourea, 1 % (w/v) CHAPS, 20 mM DTT, 0.8 % (v/v) carrier ampholytes 3-10, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 14 µM PMSF). The protein concentration was determined and aliquots of 100 µg stored at - 20 °C.
For the separation in the first dimension (IEF) the volume of the urea buffer was adjusted to 350 µl and the proteins transfered by in-gel rehydration into the IEF gels.

The serum was collected, proteins centrifuged down and washed in 1 ml TE-PMSF (1 mM EDTA, 0.1 M Tris, 14 µM PMSF, pH 7.5). Then the protein pellet was precipitated in acetone (80 % (v/v) acetone, 0.12 % (w/v) DTT) for at least 2 h at – 20 °C, pelleted by centrifugation (30 min, 14.000 rpm, 4 °C) and resuspended in urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10). The protein concentration was determined and aliquots of 100 µg stored at - 20 °C.
Prior to separation in the first dimension (IEF) the volume of the urea buffer was adjusted to 350 µl and the proteins transfered by in-gel rehydration into the IEF gels.

Intact and carious (advanced enamel or early to moderate dentinal caries) third molars were removed. External surfaces of the teeth were first cleaned with 75 % (v/v) ethanol and then mechanically to remove all the external soft tissue remnants, blood and cementum. A 2-3 mm deep groove was cut around the teeth with a 0.1 mm one-sided diamond disc, avoiding the exposure of the pulp. The cut was placed 3-4 mm apically from the cement-enamel junction in order to preserve as much of the pulp tissue coronally from the cutting line as possible, to maximize the tissue sample size of the tooth. The root was fractured off trough the cutting line with the sharp-edged pliers, and pulp was gently removed from the coronal pulp chamber, washed thrice with 1 x PBS. Total RNA was isolated according to the Trizol® protocol (GIBCO-BRL, Gaithersburg, MD, USA) and the proteins sedimented from the organic phase of the extract using isopropanol.
a. Protein separation for silver staining
The pellet was resuspended in urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10) and the protein concentration determined. Aliquots of 100 µg or 500 µg were stored at - 20 °C. For the separation in the first dimension (IEF) the volume of the urea buffer was adjusted to 350 µl and the proteins transferred by in-gel rehydration into the IEF gels.
b. Separation of Cy3 labelled proteins
For protein labelling with the fluorescence dye Cy3 the protein pellet was first precipitated with 80 % (v/v) acetone for at least 2 h at – 20 °C and pelleted by centrifugation (30 min, 14.000 rpm, 4 °C). The following steps were performed according to the manufacturer`s protocol (CyDye DIGE Fluor Labelling Kit for Scarce Samples, A. Biosciences) with slight modifications. In brief, the protein pellet was resuspended in urea buffer (8 M urea, 2 M thiourea, 4% (w/v) CHAPS, complete Mini protease inhibitor, pH 8.0), sonicated (10 min, ultrasonic bath) and mixed. After centrifugation (10 min, 11.400 rpm, room temperature) the supernatant was transferred into a new tube and the pH and protein concentration determined. If necessary the pH was adjusted to 8.0. Protein aliquots of 10 µg were adjusted to a final volume of 9 µl with urea buffer. For the labelling 2 µl (4 mM) TCEP were added, mixed and incubated for 1 h at 37 °C in the dark. Now 4 µl Cy3 dye (8 nmol) were added, mixed and incubated for 30 min at 37 °C in the dark. After that 15 µl urea buffer containing also 130 mM DTT and 2 % (v/v) carrier ampholytes 3-10 were added and mixed to stop the labelling reaction. The protein extracts were stored at -20 °C for later use.
For the separation in the first dimension (IEF) the IEF gels were incubated in 350 µl urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10) without protein. Prior separation the protein solution was applied into sample cups placed at the anodic end of the IEF gel.
For the separation by SDS-PAGE the IEF gels were equilibrated for 15 min in equilibration solution C (100 ml solution contained 10 ml 1 M Tris/HCl pH 8.0, 36 g urea, 30 ml glycerin, 2 g SDS, 0.5 g DTT, few bromphenol blue) instead of equilibration solution A and B.

The IEF is an electrophoretic method for the separation of proteins according to their charge in a specific pH gradient. Proteins are separated within an electrical field until they reach the position in the gel were their own charge is zero (isoelectric point, pI).

The protein solution was adjusted with rehydration solution to a final volume of 350 µl (for 18 cm long IEF gels). The IPG strips (e.g. pH 3–10, non linear, pH 4-7) were incubated over night in the rehydration solution to mediate the protein transfer. IEF was carried out at 20 °C with the Multiphor II system (Amersham Biosciences) under mineral oil (PlusOne Immobiline DryStrip Cover Fluid, Amersham Biosciences) for 55 kVh (500 V 500Vh, 500 V 2500 Vh, 3500 V 10 kVh, 3500 V 42 kVh; linear gradient) according to Buttner et al., 2001.

The IEF gels (18 cm) were incubated over night in 350 µl rehydration solution. Prior the separation the sample cups were placed onto the gel surface at the anodic side of the gel. The gels were overlaid with mineral oil (PlusOne Immobiline DryStrip Cover Fluid) to control the correct positioning of the sample cups. Then the protein solution (20-100 µl) was applied into the cups. IEF was carried out at 20 °C with the Multiphor II system (Amersham Biosciences) for 52 kVh (150 V 2 h, 300 V 4 h, 1500 V 14 h, 3500 V 8,5h; step gradient) according to Görg et al., 2000.
The strips were stored in aluminium foil at - 20°C or prepared for SDS-PAGE.

During SDS-PAGE the proteins are separated according to their molecular mass in a polyacrylamide gel of specific concentration. Prior to SDS-PAGE the proteins are saturated with the strong anionic detergent SDS in order to mask the own charge of the proteins, which might disturb the separation. SDS forms complexes with the proteins and "overrides" their charge.
The combination of IEF and SDS-PAGE allows the separation of proteins dependent on 2 different parameters (charge, molecular mass) and results in a two-dimensional protein pattern, which allows the analysis of thousands of protein spots within one single 2-D gel at the same time.

Prior to preparation of the gel solutions the gel caster (Amersham Biosciences) was prepared. For the separation on the second dimension the gel solutions were prepared. In order to reach an uniform separation we used also a stacking gel in addition to the slab gel. The slab gel (12 %, approx. 900 ml for twelve 25.5 x 20.5 cm gels) was prepared containing the following components: 263 ml 40 % (w/v) acrylamide for 2D-PAGE, 144 ml 2 % (w/v) Bis, 225 ml 1.5 M Tris/HCl pH 8.8, 9 ml 10 % (w/ v) SDS and 259 ml distilled water. The corresponding stacking gel (4 %, approx. 100 ml for 12 gels) contained 9 ml 40 % (w/v) acrylamide for 2D-PAGE, 4.5 ml 2 % (w/v) Bis, 25 ml 0.5 M Tris/ HCl pH 6.8, 1 ml 10 % (w/v) SDS, 60 ml distilled water. The stacking gel was stored at + 4 °C to slow the polymerization. Then 4.5 ml 10 % (w/v) APS and 0.45 ml TEMED were added to the slab gel solution and the solution casted quickly through a tube into the gel caster. Each gel was overlaid with 1 ml deionized water to reach an uniform gel surface. After 1.5 - 2 h at room temperature the slab gel was polymerised and the overlaid deionized water removed. Then 0.5 ml 10 % (w/v) APS and 0.1 ml TEMED were added to the stacking gel solution. Now the solution was applied onto the slab gel. After that the stacking gel surface was also overlaid with deionized water and the gel polymerised in 1 – 1.5 h.

		The preparation of the 1.5 M Tris/ HCl stock solution and the running temperature strongly influence the protein separation!
Click here to see the influence of the temperature on the pH in the 1.5 M Tris/ HCl solution
Click here to view the separation of low molecular mass proteins dependent on the pH and temperature We commonly use a Tris solution with a pH 8.80 calibrated at 27 °C, which reflects pH 9.22 at 12 °C. The pH in the complete slab gel solution is pH 9.18 during the running temperature at 12 °C and allows the detection of proteins with a molecular mass down to 6 kDa.

The 1-D SDS-PAGE was performed in the Mini-PROTEAN 3 Cell (Bio-Rad). Prior to preparation of the gel solutions the casting stand (Bio-Rad) was prepared with casting frames and glass plates according to the manufactors protocol and the glass plate marked 1 cm below the comb teeth. After that the gel solutions were prepared. In order to reach an uniform separation we used also a stacking gel in addition to the slab gel. The slab gel (12 %, approx. 10 ml for two 10 x 7.5 cm gels) was prepared containing the following components: 2.92 ml 40 % (w/v) acrylamide for 2D-PAGE, 1.6 ml 2 % (w/v) Bis, 2.5 ml 1.5 M Tris/HCl pH 8.8, 0.1 ml 10 % (w/ v) SDS and 2.88 ml distilled water. The corresponding stacking gel (4 %, approx. 10 ml for two gels) contained 0.9 ml 40 % (w/v) acrylamide for 2D-PAGE, 0.45 ml 2 % (w/v) Bis, 2.5 ml 0.5 M Tris/ HCl pH 6.8, 0.1 ml 10 % (w/v) SDS, 5.99 ml distilled water. Then 75 µl 10 % (w/v) APS and 10 µl TEMED were added to the slab gel solution and the solution casted between both glass plates. Each gel was overlaid with 1 ml deionized water to reach an uniform gel surface. After 1 h at room temperature the slab gel was polymerised and the overlaid deionized water removed carefully with a filter paper. Then 75 µl 10 % (w/v) APS and 10 µl TEMED were added to the stacking gel solution. Now the solution was applied onto the slab gel and the comb inserted. After 30-45 min the gels were polymerized, the comb was removed and the gel surface rinsed with deionized water.

Prior to separation in the second dimension the strips were equilibrated according to Görg et al., 2000. In brief, the IEF gels (IPGs) were equilibrated under slightly shaking for 15 min in 3-4 ml equilibration solution A (100 ml solution A contained 10 ml 0.5 M Tris/HCl pH 6.8, 36 g urea, 30 ml glycerin, 4 g SDS, 0.35 g DTT), followed by incubation for additional 15 min in the same volume of equilibration solution B (100 ml solution B contained 10 ml 0.5 M Tris/ HCl pH 6.8, 36 g urea, 30 ml glycerin, 4 g SDS, 4.5 g iodoacetamide, few bromphenol blue). The casting cassettes (containing the polymerized gels) were short washed with deionized water to remove gel pieces and put into the separation chambe. Then the stacking gels were overlaid with 1 x SDS buffer (prepared from 10 x SDS buffer: 1 liter contained 30 g Tris, 144 g glycine, 15 g SDS). Excess of equilibration solution was removed by dipping the IEF gels short into deionized water. Now the IEF gels were placed directly onto the gel surface and remaining air bubbles between both gels removed. Finally the 2-D gels were overlaid with 1 x SDS buffer and the proteins separated over night at 12 °C with 1- 2 W/ gel (14 W/ 12 gels; 500 V, 300 mA; Ettan DALT II system, Amersham Biosciences). The separation was stopped, when the bromphenol blue reached the lower gel end. If the run was not finished after over night electrophoresis the power was increased to a maximum of 4 W/ gel.

The gel cassettes were placed in the assembly module and about 120 ml and 200 ml of 1 x SDS buffer (prepared from 10 x SDS buffer: 1 liter contained 30 g Tris, 144 g glycine, 15 g SDS) were filled in the inner and lower chamber. The wells were rinsed with SDS buffer. The protein sample (up to 10 µg) solved in 1 x sample buffer (2 x sample buffer: 2 ml 0.5 M Tris/ HCl pH 6.8, 3.6 ml 86 % glycerin, 0.31g SDS, 0.124 g DTT, pinch of bromphenolblue, adjust to 10 ml with distilled water) was heated at 95 °C for 5 min. The protein sample was applied into the wells and separated (150 V, 35-60 min) until the bromphenolblue reached the lower gel end.

With a detection limit down to 1-2 ng silver staining offers the possibility to detect also low abundant proteins. After SDS-PAGE the gels were gentle shaked for 2 h in fixing solution (50 % (v/v) ethanol, 12 % (v/v) acetic acid, 0.0185 % (v/v) formaldehyde). During all following steps the gels were gentle shaken. Now the gels were washed with 50 % ethanol (3 x 20 min) to clean the gel for the staining process followed by an incubation for exact 1 min in sensitising solution (0.02 % (w/v) Na2S2O3 x 5 H2O). The gels were immediately washed with deionized water (2 x 30 s) and incubated for 20 min in silver solution (0.2 % (w/v) AgNO3, 0.028 % (v/v) formaldehyde), washed again with deionized water (2 x 30 s) to remove the remaining silver solution and incubated in developing solution (3 % (w/v) K2CO3, 0.0004 % (w/v) Na2S2O3 x 5 H2O, 0.0185 % (v/v) formaldehyde). The developing reaction was stopped by incubating the gels for 20 min in a solution containing 1 % (w/v) glycine. The gels were then washed for at least 30 min in deionized water, placed between 2 plastic foils and sealed.
Now the stained gels can be stored for years at room temperature, but should be placed in the dark to avoid fading of the protein pattern.

High purity (photographic grade) of K2CO3 is required for an optimal silver staining!

The immunoblot was performed with the Mini Trans-Blot Cell (Bio-Rad) for 10 x 7.5 cm gels or excised parts of larger gels with the same final size. Prior the transfer the cooling units were stored with deionized water at -20 °C and also about 1 liter of transferbuffer (25 mM Tris, 192 mM glycine, 20 % ethanol, adjust pH 8.1-8.4) was cooled at 4 °C. Immediately after electrophoresis the gel, membrane (PROTEAN nitrocellulose membrane, Schleicher & Schuell), filter papers and fiber pads were soaked in transferbuffer for 5-10 min. Now one fiber pad, one filter paper, the gel, the membrane, one filter paper were placed onto the gray side of the gel holder cassette. Any air bubbles between membrane and gel were removed and one fiber pad was added. The cassette was closed, placed into the electrode module and the module inserted into the buffer tank. The pre-cooled cooling units and a magnetic stirrer were placed in the module. Now the tank was placed on a stir plate and completely filled with cooled transfer buffer and the electrophoresis started (100 V, 1 h, 350 mA). After the electrophoresis the quality of the transfer was checked by staining the membrane for 30 s in Ponceau S (1 % Ponceau S, 0.1 % acetic acid) followed by short destaining with 1 x TBST (10 x TBST: 22.4 g Tris, 80 g NaCl, adjust pH 7.6, adjust volume to 1 liter). The stained membrane was placed into plastic foil and scanned. The membrane was incubated 30-60 min in 1 x TBST containing 1 % skim milk powder, washed 2 x 10 min in 1 x TBST and incubated for 1 h (or over night) in 1 x TBST containing primary antibody. After that the membrane was rinsed with 1 x TBST, washed 2 x 10 min in 1 x TBST and incubated for 1 h in 1 x TBST containing secondary antibody coupled to a specific enzyme (e.g. peroxidase). After that the membrane was rinsed again with 1 x TBST, washed 3 x 10 min in 1 x TBST and the membrane incubated for 1 min in a solution with a specific substrate for the enzyme. For this reaction we used the ECL Western blotting detection kit (Amersham Biosciences). After this incubation the membrane was placed between plastic foil and placed on X-ray films. The position of the obtained signal was compared with the pattern of the stained membrane and a silver stained 2-D gel of the same sample run in parallel with the blotted 2-D gel.

Excised gel pieces from silver stained 2-D gels separated with 100 µg or 500 µg protein were washed with 200 µl distilled water and destained with 50 µl 100 mM Na2S2O3/ 30 mM [K3Fe(CN)6] for 10 min at room temperature. The destained gel was washed twice with 200 µl destilled water, then with 200 µl of a solution containing 200 mM NH4HCO3 and 40% CH3CN, followed by dehydration with 50 µl CH3CN. The gel pieces were dried, cooled on ice and soaked with trypsin solution (20 µg/ml porcine trypsin in 40 mM NH4HCO3, 10% CH3CN and 0.5 % beta-octyl-D-glucoside). After 30 min on ice and complete rehydration the gel pieces were incubated overnight at 37 °C. Digested peptides were recovered from the gel by consecutive extraction for 10 min each in an ultrasonic bath with 20 µl 3 % TFA/ 5 % CH3CN, 0.2 % TFA/ 30 % CH3CN, 0.1 % TFA/ 50 % CH3CN, 0.1 % TFA/ 50 % CH3CN, and 0.1 % TFA/ 70 % CH3CN. Extracts were pooled, dried, and peptides directly dissolved in 5 µl matrix solution (alpha-cyano-4-hydroxy cinnamic acid, saturated solution in 40 % CH3CN, 0.1 % TFA). The samples (1 µl) were subjected to mass analyses on a MALDI-TOF mass spectrometer (Voyager DE-STR, Applied Biosystems, Foster City, CA) operating in reflector mode following parameters (20000 V acceleration potential, 200 ns delay time, 67 % grid voltage) optimized for peptide masses between 1000 and 2000 Da. Spectra were calibrated internally with trypsin peaks (842.5100, 2211.1046) and the 40 strongest monoisotopic peaks subjected to ProFound using the database NCBI. The identification of a protein was accepted if the peptides (mass tolerance 20 ppm) covered at least 30 % of the complete sequence. A sequence coverage between 30 % and 20 % or a sequence coverage below 20 % for protein fragments was only accepted if at least three main peaks of the mass spectrum matched with the sequence and the number of weak-intensity peaks was clearly reduced.

Bloom, H., Beier, H., Gross, H. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis, 8, 93-99.

Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254

Görg, A., Boguth, G., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R., Weiss, W. (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 21, 1037-1053.

Buettner, K., Bernhardt, J., Scharf, C., Schmid, R., Mäder, U., Eymann, C., Antelmann, H., Völker, A., Völker, U., Hecker, M. (2001) A comprehensive two-dimensional map of cytosolic proteins of Bacillus subtilis. Electrophoresis, 22, 2908-2935.

Meisinger, C., Sommer, T., Pfanner, N. (2000) Anal Biochem 287(2), 339-342.

O`Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem., 250, 4007-4021.