Evaluation of proteins

3.8.1 Extraction of protein from tissues

Tissue samples stored at -80°C were weighed, placed in 5 ml plastic tubes and homogenized by 23500 rpm in protein extraction buffer with a tissue homogenizer for 1 minute. For 20 mg tissue, 500 µl extraction buffer were used. The homogenizer was cleaned after each sample with PBS.

5x Laemmli buffer: 1M Tris (pH 6.8) 65.5 ml glycerol 100 ml 0.5M EDTA (pH 8.0) 2 ml SDS 20 g bromphenol blue 0.1 % bidistilled water up to 200 ml

protein extraction buffer: 1M Tris (pH 7.5) 2ml Triton X-100 2ml 5x Laemmli buffer 20ml bidistilled water 76ml

The samples were stored on ice, transferred to 1.5 ml centrifuge tubes, incubated at 95°C for 5 min, chilled on ice for 10 min and centrifuged at 13000 rpm, 4°C, for 5 min. 10µl were removed for determination of protein concentration and samples were stored at -20 °C.

3.8.2 Determination of protein concentration

The protein content of samples was estimated by the bicinchoninic acid (BCA) protein assay. A set of protein standards of known concentrations was prepared by serially diluting a bovine serum albumin (BSA) stock solution (4 mg/ml) in PBS. 50 µl of the standards and of the samples of unknown concentration (diluted 1:10 in PBS) were pipetted into 96-well plates and 200 µl of a mixture of bicinchoninic acid and 4% CuSO4 (50:1) were added to each well and

nm. A standard curve was prepared by plotting the absorbance of standards versus their protein concentration. The protein concentration of the samples was determined using this standard curve.

3.8.3 SDS-Polyacrylamide gel electorphoresis (SDS-PAGE)

20 µg of each protein sample were pipetted into centrifuge tubes and filled up with 1x Laemmli buffer to the same volume. The samples were incubated at 95°C for 5 min and chilled on ice. In some experiments 2-mercaptoethanol was added to the protein samples resulting in a final concentration of 5 %.

The proteins were separated using the Mini Protean® 3 cell system (Bio-Rad). The separating gel (12% acrylamide) was prepared in an Erlenmeyer flask under continuous agitation and poured into the gap between the glass plates of the system, leaving space enough for the stacking gel (about 2.5 cm). The separating gel was overlaid with bidistilled water to ensure an even surface.

separating gel (12 %): bidistilled water 3.35 ml 1.5 M Tris (pH 8.8) 2.5 ml 30% acrylamide 4.0 ml 10% SDS 100 µl 10% ammonium persulfate 50 µl Temed 5 µl

After complete polymerization (45 min) the water was discarded and the stacking gel (5% acrylamide) was prepared in the same way and loaded on the top of the separating gel. The comb was inserted taking care to not trap air bubbles under the teeth.

stacking gel (5 %): bidistilled water 7.0 ml 0.5 M Tris (pH 6.8) 1.25 ml 30% acrylamide 1.5 ml 10% SDS 100 µl 10% ammonium persulfate 100 µl Temed 5 µl

After complete polymerisation (30 min) the comb was removed and the plates were mounted in the electrophoresis apparatus which was filled with SDS-page electrophoresis buffer.

SDS-PAGE electrophoresis buffer: Tris 30.3g glycine 144g SDS 10g bidistilled water up to 1l

Samples were loaded and the electrophoresis was performed initially at 100V for 10 min and then at 140V until the bromophenol blue left the separating gel at the bottom. A molecular weight standard (PageRulerTM Prestained Protein Ladder #SM0671, MBI Fermentas etc.) was pipetted into the first slot for estimation of the protein size.

3.8.4 Electrophoretic blotting

The gel was removed from the electrophoresis chamber and the separated proteins were transferred to a PVDF membrane (Millipore) by semidry blotting in an electroblotter (Bio- Rad). One sheet of gel blotting paper (Bio-Rad) with the same size of the gel was soaked in transfer buffer stacked on the bottom of the electrode, and squeezed with a pipette to remove air bubbles. The PVDF membrane and then the gel were placed exactly over the paper and were covered with another blotting paper. The upper electrode was positioned and the system connected to a power supply (Bio-Rad). The transfer took place for 55 min at 15 mA for all gels.

10x transfer buffer: Tris 58.2g glycine 29.2g SDS 3.7g bidistilled water up to 1l

Methanol was added to the freshly prepared 1x solution to a final concentration of 20%.

After the transfer the membrane was unambiguously labelled with a pen, stained with Ponceau S solution for 5 min, and rinsed with distilled water. Air-dried membranes were stored at 4°C.

3.8.5 Detection of the antigen

The membrane was rinsed for 1 min in methanol and than rinsed 3 times for 5 min in TBS-T buffer on a seesaw at RT.

10x TBS buffer: Tris 30 g NaCl 80 g bidistilled water up to 1 l

TBS-T buffer: 1x TBS

0.1 % Tween 20

The membrane was incubated in blocking solution at RT for 60 min. blocking solution: 5 % instant skimmed milk powder

TBS-T

After blocking, the primary antibody was diluted in blocking solution or in 5% BSA diluted in TBS-T. When BSA was employed, the membrane was washed 4 times for 5 min at RT in TBS-T before being incubated with the antibody. The incubation took place over night on a seesaw at 4°C. At the next day the membrane was washed 3 times for 5 min at RT with TBS- T. Incubation with the secondary antibody diluted in blocking solution was carried out for 1 hour. After incubation the membrane was washed 3 times for 5 min at RT. Detection was performed by incubating the membrane with 2 ml of the ECL Western blotting detection reagent (RPN 2106) (GE Healthcare). The membrane was sealed under plastic and exposed to an ECL film (GE Healthcare).

primary antibodies: goat anti-amylase (C-20) sc-12821 (Santa Cruz, Heidelberg, Germany) diluted 1:1000 in 5% milk in TBS-T

rabbit anti-BAX (554106) (BD) diluted 1:1000 in 5% milk in TBS-T rabbit anti-Bcl-2 (#2876) (Cell Signaling) diluted 1:1000 in 5% BSA in TBS-T

rabbit anti-Bcl-xL (#2762) (Cell Signaling) diluted 1:1000 in 5% BSA in TBS-T

rabbit anti-c-Jun (#9162) (Cell Signaling) diluted 1:500 in 5% BSA in TBS-T

rabbit anti-Phospho-c-Jun (#9164) (Cell Signaling) diluted 1:500 in 5% BSA in TBS-T

rabbit anti-Phospho-SAPK/JNK (#9251) (Cell Signaling) diluted 1:1000 in 5% BSA in TBS-T

rabbit anti-SAPK/JNK (#9252) (Cell Signaling) diluted 1:1000 in 5% BSA in TBS-T

mouse anti-p-ERK (E-4) sc-7383 (Santa Cruz) diluted 1:1000 in 5% BSA in TBS-T

rabbit anti-ERK 1 (C-16) sc-93 (Santa Cruz) diluted 1:1000 in 5% milk in TBS-T

rabbit anti-ERK 2 (C-14) sc-154 (Santa Cruz) diluted 1:1000 in 5% milk in TBS-T

rabbit anti-Phospho-eIF2α (#9721) (Cell Signaling) diluted 1:1000 in 5% BSA in TBS-T

rabbit anti-eIF2α (#9722) (Cell Signaling) diluted 1:1000 in 5% BSA in TBS-T

rabbit anti-Phospho-p38 MAPK (#4631) (Cell Signaling) diluted 1:1000 in 5% BSA in TBS-T

rabbit anti-p38 MAPK (#9212) (Cell Signaling) diluted 1:1000 in 5% BSA in TBS-T

mouse anti-PARP (556362) (BD) diluted 1:1000 in 5% milk in TBS-T goat anti-betacellulin (R&D, Germany) diluted 1:1000 in 3% milk in TBS-T

secondary antibodys: sheep anti-mouse HRP (NA 931V) (Amersham, Freiburg, Germany) diluted 1:1000 in 5% milk in TBS-T

donkey anti-rabbit HRP (NA 934V) (Amersham) diluted 1:1000 in 5% milk in TBS-T

donkey anti-goat HRP (705-035-003) (Jackson Immuno Research) diluted 1:2000 in 5% milk in TBS-T

rabbit anti-goat HRP sc-12821 (Santa Cruz) diluted 1:5000 in 5% milk in TBS-T