Protein biochemistry

2.15.1. Preparation of whole cell protein lysates

Cells were harvested by trypsinization (see 2.12.1.), resulting cell pellets were washed twice with 50 ml 1x PBS, and cells were diluted in an appropriate volume of 1x PBS. Cells were disrupted by rotational freezing in liquid nitrogen followed by thawing on ice for three times.

The cell lysates were centrifuged to sediment the residual cell debris (10 min, 16000 x g, 4 °C).

Resulting supernatant was transferred into a new reaction tube. Photometric quantification of protein concentrations was performed by the Bradford method (Bradford, 1976) using the Roti^®-Quant Kit according to manufacturer’s instructions.

2.15.2. Immunoprecipitation (IP)

HEK293A cells were transiently transfected with Attractene transfection reagent (see 2.12.3.) and harvested 24 h after transfection by trypsinization (see 2.12.1.). The resulting cell pellets were diluted in 1 ml IP lysis buffer, containing freshly added 1 mM PMSF and protease inhibitor (Complete ULTRA Tablets Mini EDTA free EASYpack), and were incubated on ice for 1 h. Cell suspension was centrifuged (10 min, 16000 x g, 4 °C) before the protein concentration

44 in the supernatant was determined by the Bradford method using the Roti^®-Quant Kit. For later control purpose an aliquot containing 65 µg protein was taken from the supernatant (input), mixed with 9 % Laemmli buffer and stored at -20 °C. The remaining supernatant was divided into half (myc IP and control IP). To increase the amount of protein for the co-immunoprecipitation, total protein from one 175 cm² culture flask with confluently grown, untransfected HEK293A cells was added to a final volume of 4 ml IP lysis buffer. Precipitation of XPG(mut)mycHisprotein was performed over night on a rotating wheel at 4 °C by adding an αmyc antibody in a dilution of 1:1000 to the myc IP sample. Control IP was performed under similar conditions using an αmouse IgG control antibody. Next day, 50 µl of a 1:1 mixture of AgaroseA beads (50 % slurry) and AgaroseG+ beads (50 % slurry), equilibrated with IP lysis buffer, were added to each sample and samples were further incubated for 2 h. The antibodies bind to the agarose beads and, subsequently, the antibody-protein complexes are immobilized on the beads. Beads with bound protein complexes were washed five times with IP lysis buffer.

Beads were resuspended in 30 µl 9 % Laemmli buffer and boiled for 5 min at 96 °C. The 9 % Laemmli buffer contains 10 % (v/v) β-mercaptoethanol reducing disulfide bridges of proteins needed for protein unfolding. Boiling the samples results in the dissolution of protein-antibody-agarose-complexes. Subsequently, the proteins can be separated by size with SDS-PAGE (see 2.15.3).

2.15.3. Horizontal SDS-PAGE and Western Blotting

Dilution and boiling (5 min at 96 °C) of protein samples in Laemmli buffer containing SDS (sodium dodecyl sulphate) results in denaturated and negatively charged proteins. Therefore, samples can be separated by size in an electric field using polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970).

Protein samples were analyzed by horizontal PAGE followed by immunoblotting. For SDS-PAGE the Amersham™ ECL™ electrophoresis system with a precast 4 % to 12 % polyacrylamide gradient gel was used according to manufacturer’s instructions. Protein transfer from the polyacrylamide gel to a nitrocellulose membrane was performed applying the wet-blot method using an XCellII Blot Module at a voltage of 25 V and a current of maximal 300 mA for 2.5 h at 4°C. Afterwards, free protein binding sites were saturated by incubation of the membrane for 30 min at room temperature in blocking buffer (blocking buffer was used for each antibody according to manufacturer’s instructions). Incubation with the specific antibodies was performed rocking over night at 4 °C. All following steps were performed at room temperature using solutions from the WesternBreeze Chemiluminescent Immunodetection Systems (anti

45 mouse or anti rabbit). The membrane was washed four times for 5 min with washing buffer before incubation with the secondary antibody was performed for 30 min. Afterwards, the membrane was washed four times for 5 min with washing buffer again and rinsed with aqua bidest before chemiluminescent substrates were added. The resulting chemiluminescent signal was detected with a luminescent image analyzer LAS-4000.

2.15.4. Immunofluorescence (XP protein recruitment kinetics)

Fibroblast cells were seeded at a density of 2x10⁴ cells per well on glass cover slips in 24-well-plates. Next day, medium was taken from the cells, kept for later use, and cells were washed twice with 1x PBS. 1x PBS was removed and cells were covered with 8 µm isopore polycarbonate membrane before they were irradiated with 100 J/m² UVC using an ultraviolet crosslinker with 254 nm UV light bulbs. The medium was given back to the cells and cells were further incubated for time intervals of the kinetic (6 min, 15 min, 30 min, 3 h, 6 h, 24 h). Cells were washed three times with 1x PBS to remove the media before they were fixed with 300 µl 3.7 % PFA for 15 min at room temperature. Cells were washed again three times with 1x PBS to remove remaining PFA before they were permeabilized with 0.1 % Triton-X-100 in 1x PBS for 15 min at room temperature. Additional threefold washing with 1x PBS was performed and cells were blocked with 20 % FCS in 1x PBS for 20 min at room temperature. Again, cells were washed threefold with 1x PBS before incubation with an antibody directed against one of the XP-proteins (XPA, XPB, XPC, ERCC1, or XPG) in a 1:50 dilution for 1 h at 37 °C was carried out.

Afterwards, cells were washed three times with 1x PBS containing 0.05 % Tween-20 (1x PBS-Tween) and incubated with the secondary αrabbit antibody conjugated with DyLight488 in a 1:400 dilution for 1 h at 37 °C.For single staining against a XP protein, the glass cover slip was mounted with mounting medium for fluorescence containing DAPI and stored at 4 °C and protected from light until further use.

For a double staining of a XP protein and a photoproduct or single staining of a photoproduct the DNA had to be denaturated to become accessible for the antibody directed against the DNA photoproduct. Denaturation was performed by incubation with 2 M HCL for 20 min (double staining) or 30 min (single staining) at room temperature. After that, cells were washed three times with 1x PBS-Tween and then incubated with either CPD antibody in a 1:1000 or with 6,4PP antibody in a 1:500 dilution for 30 min at 37 °C. Cells were washed threefold with 1x PBS-Tween and incubated with secondary αmouse antibody conjugated with Dylight594 in a 1:500 dilution for 1 h at 37 °C. Cells were washed three times with 1x PBS and glass cover slips were subsequently mounted with mounting medium containing DAPI. Digital

46 images were taken with an Axio Imager.M1. For quantification at least 100 nuclei were counted for positive staining (percent positive stained nuclei).

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