2 MATERIALS AND METHODS
2.5 Biochemical methods
2.5.1 SDS-‐PAGE
SDS-‐PAGE was performed to separate proteins according to their molecular weight under denaturing conditions by one-‐dimensional SDS-‐Polyacrylamide gel electrophoresis (SDS-‐PAGE), originally developed by Laemmli (1970). Gels used in this study contained 0.1% SDS and 4% acrylamide for the stacking gel and 12.5-‐17%
acrylamide for the separating gel (depending on the size of proteins to separate).
Acrylamide stock solution used to prepare gels contained 30%
acrylamide/bisacrylamide (37.5 : 1). Samples were mixed with SDS loading buffer and incubated at 25 °C for 15 min prior to loading. Electrophoresis was performed in custom-‐made midi gel systems (250 V, 30 mA per gel). As a standard, SDS-‐PAGE standard Broad Range (Biorad) was used.
2.5.2 Urea-‐SDS-‐PAGE
For better separation and resolution in the low molecular range (below 10 kDa), SDS-‐PAGE gels, containing urea were used. Handling and electrophoresis conditions were basically identical to SDS-‐PAGE gels. The resolving gel contained 5.4 M urea, 0.09% SDS, 17.5% acrylamide, 0.23% bisacrylamide. The stacking gel contained 5.5%
acrylamide/0.07% bisacrylamide, 0.12% SDS, 3.6 M urea. The running buffer was composed of 50 mM Tris, 192 mM glycine, 0.1% SDS. Electrophoresis was performed for 4-‐5 h.
2.5.3 BN-‐PAGE
For separation of native protein complexes, Blue Native polyacrylamide gel electrophoresis (BN-‐PAGE) was basically performed as described earlier (Schägger &
Jagow 1991; Wittig et al. 2006). Prior to the gel run, mitochondria were solubilized in digitonin solubilization buffer, containing 1% digitonin, at a concentration of 1mg/ml.
To ensure proper solubilization, mitochondria were re-‐isolated by centrifugation (14000 rpm, 10 min, 4 °C) and resuspended by pipetting up and down 12 times using a small tip. After incubation of the samples for 30 min on ice, insoluble material was removed by centrifugation (14000 rpm, 15 min, 4 °C). The supernatant was mixed with BN loading dye to a final concentration of 1x.
4-‐10% or 4-‐13% gradient gels with 4% stacking gel in a Hoefer gel system, equipped with a cooling device to allow electrophoresis at 4 °C were used in this study. All buffers were precooled to 4 °C and gel loading was performed in a cold room at 4 °C.
Electrophoresis was performed with anode buffer, containing Coomassie at 200 V and 15 mA per gel for 2 h. Anode buffer was replaced by anode buffer without Coomassie and electrophoresis was continued at 600V and 15 mA per gel (3-‐4 h). Replacement of the anode buffer was omitted if the gel was not intended for Western blotting. Following the gel run, the gel was either stained with Coomassie or subjected to Western blotting.
As a standard, the HMW calibration kit (GE Healthcare) was used.
For analysis of steady state protein levels of structural subunits of respiratory chain complexes, 15-‐20 µg of mitochondria were loaded per lane. For analysis of steady state levels of less abundant proteins (e.g. COX assembly factors), 50-‐70 µg of mitochondria were loaded per lane. For analysis of elution of protein complex isolations, the amount loaded per lane varied, but usually corresponded to a starting material between 250-‐500µg of mitochondria.
2.5.4 Determination of protein concentrations
Protein concentrations were determined by Bradford analysis (Bradford 1976) using Roti-‐quant (Roth) according to the manufacturers specifications with bovine IgG (Biorad) as a standard. Absorbance of the protein solutions was measured using a BioPhotometer (Eppendorf) at 595 nm. Protein concentrations were calculated based on the IgG standard.
2.5.5 Yeast whole cell extracts
For analysis of proteins on a whole cell level, whole cell extracts were prepared essentially as described earlier (Yaffe & Schatz 1984). In detail, yeast cells were grown in YPGal overnight. After determination of OD600, 2 OD600 were isolated by centrifugation (14000 rpm, 1 min at RT). The cells were resuspended in dH2O, 148 µl 2 M NaOH and 12 µl β-‐mercaptoethanol were added and the mixture incubated on ice for 10 min. Trichloracetic acid (TCA) was added to a final concentration of 7% and incubated on ice for 10 min. Precipitated material was pelleted by centrifugation (2 min, 14000 rpm, 4 °C) and the pellet resuspended in SDS sample buffer. The pH of the whole cell lysate was adjusted to a neutral pH (indicated by the blue colour of the SDS loading buffer) by titration with 1 M Tris (pH 11.5). Before the samples were subjected to SDS-‐
PAGE, debris was removed by centrifugation (14000 rpm, 1 min at RT).
2.5.6 Western Blotting
After separation by polyacrylamide gelelectrophoresis, proteins were transferred and immobilized onto PVDF membranes (Millipore) using semidry blotting chambers (Peqlab). PVDF membranes were pre-‐activated by short incubation in MeOH. The membrane was subsequently soaked in blotting buffer, together with gels and Whatman paper. After assembly of the blotting sandwich, transfer was performed at 25 V and 250 mA for 90 min (following SDS-‐PAGE) or for 120 min (following BN-‐PAGE and Urea-‐SDS-‐
PAGE).
2.5.7 Coomassie staining
Proteins on PVDF membranes or in polyacrylamide gels were stained with Coomassie Brilliant Blue R-‐250 for 2 min (membranes) or for 1h (gels). After staining, marker bands were labeled on the membrane and membranes were destained for 5 min, gels for up to 1 h. Additionally, PVDF membranes were completely destained in MeOH.
2.5.8 Immunodecoration of proteins on PVDF membranes
Destained PVDF membranes were blocked using 5% milk powder in TBS-‐T for 1 h at room temperature or over night at 4 °C. Following blocking of non-‐specific interaction sites, membranes were incubated in primary antibody solution (in 5% milk in TBS-‐T likewise) for 1h , washed 3 times for 10 min in fresh TBS-‐T. Subsequently, membranes were incubated in appropriate secondary antibody, coupled to HRP (solution prepared 1:5000-‐ 1:10000 in 5% milk in TBS-‐T) for 1h, followed by 3 washes for 15 min in TBS-‐T.
All incubation and washing steps were performed at room temperature and under agitation. For detection and visualization of antibody-‐protein complexes, enhanced chemiluminescence reagent (GE Healthcare) were added to the membranes and signals were detected on medical X-‐ray films (Foma).
2.5.9 Detection of radiolabeled proteins by autoradiography
For detection of radiolabeled proteins, SDS-‐ or BN-‐PAGE gels were dried in a vacuum gel drier at 65 °C for 2 h. Dried gels were exposed to Storage Phosphor Screens (GE Healthcare) for a few hours up to several days and signals were digitized using Storm820 scanner (GE Healthcare).
2.5.10 Steady state protein analyses
For steady state analysis of mitochondrial proteins by SDS-‐PAGE, mitochondria were diluted to different concentrations so that identical volumes could be loaded per gel lane. Following SDS-‐PAGE, various mitochondrial proteins from different compartments were analysed by Western blotting.
For analysis of steady state protein levels by BN-‐PAGE, 15-‐20 µg of solubilized mitochondria were loaded per lane, if structural subunits of respiratory chain complexes were assessed. For analysis of steady state levels of less abundant proteins (e.g. COX assembly factors), 50-‐70 µg of mitochondria were loaded per lane.