Sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot 73

of protein lysates, generated either by whole cell or fractionization procedures, were mixed with loading buffer containing sodium dodecyl sulfate (SDS), diluted in ddH₂O and heated to 95 °C for 5 min. As a consequence of heating and binding of SDS to hydrophobic regions of proteins, denaturation and charge-masking of proteins is triggered, respectively.

Subsequently, protein samples can be separated according to their size by segregation through SDS polyacrylamide gel electrophoresis (PAGE) either directly, or stored at -80 °C until further processing. Stacking gels were used to accumulate proteins and compensate volume differences before the proteins were separated through separation gels with according pore sizes. Stacking, as well as separation gels, were prepared according to Table 15 using 10 % gels for ATM signaling experiments and 15 % gels for all experiments involving TPT1. The separation gel was poured into the gel chamber until approximately 60–70 % of the total volume was covered and overlayed with propan-2-ol. After polymerization of the gel, the propan-2-ol was discarded and the gel chamber filled up with stacking gel.

Immediately after, the comb was inserted with care to form even sample slots. After

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polymerization of the stacker, the gel was stored at 4 °C covered in moist paper towels for a few days or used straight away.

Before usage, the comb was removed and the sample slots were washed with ddH₂O. The gel was then transferred to the electrophoresis chamber, which was filled with 1x GTS (Table 11). The electrophoresis was run at room temperature at 80 V until the samples reached the separation gel. If the samples moved evenly through the gel, the voltage was then increased to 120 V. Otherwise it was either slightly increased or not changed. The electrophoresis was stopped just before the dye front ran out.

Table 15 Composition of western blot gels

Separation gel 10 % Separation gel 15 %

Stacking gel

40 % Acrylamide 5 ml 7.6 ml 1.28 ml

2 % Bis-acrylamide 1.32 ml 1.88 ml 0.7 ml

H₂O 6.12 ml 4 ml 5.5 ml

20 % SDS 100 μl 100 µl 50 μl

1M Tris-Cl (pH 8.7) 7.52 ml 7.52 ml –

0.5M Tris-Cl (pH 6.8)

– – 2.46 ml

TEMED 20 μl 20 μl 40 μl

10 % APS 100 μl 100 μl 80 μl

In order to enable access of specific antibodies to the proteins separated by SDS-PAGE, the wet immunoblot (tank blot) technique was applied using a nitrocellulose membrane. For the transfer onto the membrane, the gel was taken out of the electrophoresis chamber and placed on the blotting module in the following order: blot module with negative terminal, sponge, two Whatman papers, gel, nitrocellulose membrane, two Whatman papers, sponge and blot module with positive terminal. Everything was prepared in 1x carbonate buffer and placed in the blotting chamber, which was filled with 1x carbonate buffer to the maximum.

The transfer was carried out at 4 °C for three hours at 35 V.

To check whether the transfer was successful, the membrane was removed from the blotting module and a Ponceau S staining was performed, which unspecifically stains abundant proteins enabling estimation of transfer completeness⁵⁰⁴. The following steps were carried out at room temperature, if not stated otherwise, and while being gently shaken. The membrane was once washed with 1x PBS and incubated with Ponceau S solution for a few minutes, until the protein lanes were clearly visible. The staining solution was then discarded and the membrane washed with ddH₂O to remove the stain. Afterwards, the membrane was blocked one hour in blocking solution (5 % skim milk powder in PBS-T) and then incubated with primary antibody (diluted in fresh blocking solution) overnight at 4 °C, to allow marking of target proteins⁵⁰⁵. Subsequently, the antibody was removed and stored at -20 °C for following usage. The membrane was washed four times with sufficient 1x PBS-T for ten minutes each, and the secondary antibody, also diluted in blocking solution, was added for an incubation time of two hours. The membrane was again washed four times with sufficient 1x PBS-T for ten minutes. For detection of the secondary antibody, which was

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coupled to horseradish-peroxidase and also enables amplification of the signal, the membrane was incubated with enhanced chemiluminescence (ECL) solution, based on luminol, according to the manufacturer’s instructions. The peroxidase catalyzes the oxidation of luminol, which subsequently emits light that can be detected by photographic density of X-ray films. If the targeted protein was ß-actin, the secondary antibody was detected with self-prepared luminol, due to the high abundance of the protein resulting in strong photographic density. After incubation with the detection solution, the membrane was shrink-wrapped in a plastic foil and the protein signals detected by light sensitive films in the dark room. The exposure time depended on the signal strength and was varied from a few seconds for ß-actin and TPT1 to half an hour for phospho-antibodies. The films were subsequently scanned and analyzed with Image J.

3.2.9 Immunocytochemistry and pre-extraction

Immunocytochemistry, - or more precisely - immunofluoresence analyses were performed to provide information on presence of proteins in intact cells and simultaneously collect data on their localization. Proteins were marked with primary antibodies analogous to the immunoblot technique and fluorescence-coupled secondary antibodies were used to enable amplification and visualization of the signal with a fluorescence microscope. For detection of irradiation-induced foci by immunofluorescence, cells were seeded and grown on cover glasses in six-well plates (Nunc) one day prior to IR treatment. Depending on the experimental setting, cells were washed three times in 1x PBS and fixed at time points ranging from 1 h to 48 h, by treatment with 3 % (w/v) formaldehyde, 2 %(w/v) sucrose in PBS for 10 min. Permeabilization was performed with 0.2 %(v/v) triton X-100 in PBS, in order to allow penetration of the cell membrane by antibodies. Primary antibodies against the target proteins were incubated in 2 % (w/v) normal goat serum (Dianova) for 2 h. After PBS washing, cells were incubated with Alexa Fluor Anti-mouse IgG 488 (Invitrogen, #A11018) and Alexa Fluor Anti-rabbit IgG 546 (Invitrogen, #A11071) or FITC-conjugated Anti-mouse IgG antibody (Zymed, #62-6511) and Alexa Fluor Anti-rabbit IgG 488 (Invitrogen, #A11070) for 2 h, respectively. DNA was counterstained with DAPI (Invitrogen) and cover classes were mounted onto slides with Pro-Long® Gold (Invitrogen) and dried overnight before evaluation by fluorescence microscopy. For pre-extraction of proteins, which are abundant in the cytoplasm, cells were subjected to detergent extraction with pre-extraction buffer containing 10 mM Hepes (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA and 0.5 % triton X-100 in PBS for 5–10 min to remove the majority of non–chromatin-bound proteins before fixation and immunostaining, in order to enable a noisefree view of the nuclear forms of these proteins²⁶⁵.

3.2.9.1 Fluorescence micoscropy and confocal laser scanning microscopy

Images were taken by Leica DMI6000B microscope (63x–100x magnification) or, for a more detailed inspection, using an Olympus confocal microscope FV 1000 (60× magnification), Table 16. Confocal microscopy is a form of light microscopy which enables depiction of three-dimensional structures by scanning different levels of a sample with a laser, which excites fluorescence molecules and produces very high resolution images⁵⁰⁶. Table 16 shows details about microscopes, objectives, and corresponding wavelengths used for excitation, as well as applied software for data processing. γH2AX and 53BP1 foci were counted manually with Image J software on pictures in an average of 100 cells after siRNA treatment

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and/or IR for each of at least two independent experiments, independent from the cell-cycle phase. Cells with apoptotic morphology or cells with intensely and completely stained nucleus were excluded from the counting process. Colocalisation was defined as yellow stained dots in overlay pictures generated with confocal microscopy. In general, the colocalization rate in experiments described in subsection 4.1.3 was very low, but at least 10 colocalization-positive cells were examined and counted manually in control cells and at least 15 cells in irradiated samples, respectively. RAD51 foci (see Supplementary figure S-25 and Supplementary figure S-27) were counted directly under the Leica fluorescence microscope using the 100x oil objective.

Table 16 Fluorescence microscopy devices and parameters

Microscope Objective with numeric

Oil immersion (60x, NA: 1.35) 532nm, 559nm, 655nm

FluoView1000