Ettan ipgphor

Isoelectric focusing was carried out by using the ETTAN IPGPhor apparatus (GE Healthcare) in Immobiline Dry Strip holders followed by approximately 750 μg protein loaded in ready strip. Briefly, a 24 cm pH 4-7 strip (GE Healthcare) was hydrated, focused, then equilibraten twice in equilibration buffer for 15 min. The IEF strips were placed on top of the 12.5% homogeneous polyacrylamide gels (26 cm-w 20 cm-h 1.0 mm-thick) in an Ettan DALT12 (GE Healthcare) gel apparatus. After the second-dimensional separation finish, the SDS-polyacrylamide gels were fixed and stained with silver nitrate. Gels were scanned on ProXpress CCD scanner (Perkin-Elmer). The images were then captured and analyzed by the Image Master 2D platinum 5.0 software, including background adjustment, landmark annotation, protein spot matching and points of difference analysis. At least three independent experiments were conducted for each group.

Each mitochondrial protein was loaded onto Immobiline^™ Drystrip immobilised pH gradient (IPG) strips (13 cm, pH 3–10 NL, GE healthcare^®) using the passive rehydration method for 12 h. Isoelectric focussing was performed using Ettan IPGphor (GE healthcare^®) at 20 °C, and the gel strips were equilibrated with the equilibration buffer twice for 15 min each, as described previously^{51 (link)}. The second dimension run was performed as follows. Each equilibrated gel strip was loaded onto a 12.5% sodium dodecyl sulphate polyacrylamide gel, sealed with the agarose sealing solution and supplied with current, using a vertical slab gel electrophoresis unit (SE 600 Chroma Hoefer^®), of 15 mA/gel for 30 min and then with a current of 30 mA/gel until the bromophenol blue reached the bottom of the gels. Gels were fixed with 10% acetic acid in 40% ethanol for 2 h, stained with the Flamingo^™ Fluorescent gel stain (Bio-Rad^®) for 18 h and washed with distilled water. 2DE was performed in triplicate.

Unless otherwise stated, chemicals used in this study were purchased from Sigma Chemical. Cultured MSC cell suspensions in lysis buffer were sonicated for 30 seconds on ice for 5 times and the soluble fractions were collected by centrifugation at 15,000 rpm for 15 min at 4°C. Prior to isoelectric focusing, IPG ReadyStrip 18 cm pH 3–10 (Bio-Rad Laboratories, Inc.) were rehydrated at room temperature for 16 hr in a lysis buffer containing 200 µg of cellular lysates. Isoelectric focusing was performed at 18°C with a current limit of 50 µA/strip. Voltage was increased progressively to a total of 60 kVhr: 1 hr at 100 V, 1 hr at 500 V, 2 hr at 1,000 V, 2 hr at 4,000 V and 10,000 V until the final voltage was reached. The focusing apparatus was an Ettan IPGphor (GE Healthcare Life Sciences). IPG strips were equilibrated for 15 min by gentle shaking in 6M urea, 2% SDS, 5 mM tributyl phosphine, 1M Tris-HCL, and 30% glycerol. Vertical SDS gradient slab gels (12.5%, 18 cm) were used in the second dimension of electrophoresis. The second-dimension gels were overlaid with a solution containing 0.5% agarose, 1 M Tris-HCl, 0.1% SDS, and a trace of bromophenol blue. Electrophoresis was conducted in SDS PAGE gel running buffer at 18 mA per gel at 18°C. The gels were fixed and stained using mass spectrometry-compatible silver stain kit (Thermo Fisher Scientific Inc.).

We used isoelectric focusing to fractionate our TMT-10plexes, and thereby reduce the complexity of the proteome. Specifically, we applied the recently developed, high resolution, isoelectric focusing method (HiRIEF) [93 ]), with an immobilized pH gradient of 3.7 to 4.9 (kindly provided by GE healthcare, Uppsala, Sweden). The TMT pooled sample (390 μg) was applied to the HiRIEF strip and run on an Ettan IPGphor (GE Healthcare) until at least 100 kVh had been reached (around 24 h). The fractionated sample was extracted from the gel strip in an automated manner, to yield 72 individual fractions. These fractions were then injected separately on a Q Exactive mass spectrometer (see section 2.6.5). This procedure was previously described in more detail [93 ].

Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) was carried out after analytical gels labeling WT and mta chloroplast proteins with Cy2 (blue), Cy3 (green) and Cy5 (red) fluorescent dyes (5 nmol Cyanine Dye DIGE Fluor mimimal Dye labeling kit, GE, USA). Preparative gels staining with coomassie blue G-250. All steps were done according to the manufacturer’s instructions. The experimental strategy is shown in S1 Fig. Three biological replicate WT samples (50 μg each) were labeled with Cy3 (one sample) and Cy5 (two samples), and the mta samples (50 μg each) were labeled with Cy3 (two samples) and Cy5 (one sample). The labeled samples were combined and separated on 2-DE gels together with the internal standard (IS), which was prepared by mixing 25 μg WT and 25 μg mta samples and labeling with Cy2. Labeling reactions were carried out according to the manufacturer’s instructions. Isoelectric focusing (IEF) was performed using Ettan IPGphor according to GE Healthcare operating manual and a previously described method [37 (link)]. All gels were scanned using a scanner (GE Healthcare, USA) according to the manufacturer's protocol. The abundance of each protein spot in the scanned images was quantified using Image Master Platinum 7.0 software (GE Healthcare, USA).

Labelled samples were used for in-gel rehydration overnight at room temperature using 24 cm Immobiline DryStrips with pH 4–7(ReadyStrip IPG Strip, Bio-Rad) for the LAP samples. The HAP fractions were processed in 13 cm linear gradient strips in the same pH range(GE Healthcare). Rehydrated strips were focused using an Ettan IPGphor(GE Healthcare). Following isoelectric focusing, each strip was equilibrated with a reducing equilibration buffer(6 M urea, 50 mM Tris-HCl pH 8.8, 30%(v/v) glycerol, 2%(w/v) SDS, 1%(w/v) DTT) for 15 min followed by equilibration with and alkylation buffer(6 M urea, 50 mM Tris-HCl, pH 8.8, 30%(v/v) glycerol, 2%(w/v) SDS, 4.8%(w/v) IAA) for 15 min. The strips were then placed on top of 12% SDS-PAGE gels and sealed with an agarose sealing solution(25 mM Tris, 192 mM glycine, 0.1% SDS, 0.5%(w/v) agarose, 0.02% Bromophenol blue). Separation of the protein in the second dimension was carried out in an Ettan Dalt-six electrophoresis tank(GE-Healthcare) in a 12% SDS-polyacrylamide gels. Resulting gels were scanned using a Pharos FX System(Bio-Rad) at 100 μm resolution.

For 2-D electrophoresis, mouse brains at 3-month-old from Efhc1^−/− mice (N = 3) were homogenized in 8 M Urea, 2% CHAPS, 2% Dithiothreitol (DTT), 1% IPG buffer, pH 3–10 NL (GE Healthcare), and Bromophenol blue (BPB). First dimensional isoelectric focusing (IEF) was carried out on an Immobiline DryStrip pH3-10NL, 7 cm (GE Healthcare) using an Ettan IPGphor (GE Healthcare). Each strip was rehydrated for 12 h with sample lysate (0.1 µg). Isoelectric focusing (IEF) was then carried out. Strips were subjected to a two-step equilibration (6 M Urea, 2% SDS, 30% glycerol, BPB) in 0.5% DTT and 4.5% iodoacetamide (nacalai tesque) buffers before proceeding to SDS-PAGE. Proteins were separated for the second dimension on 5–20% gradient SDS–polyacrylamide gel (Super Sep Ace, Wako pure reagents). After 2-D electrophoresis was terminated, one gel was stained in a solution containing 0.1% Coomassie Brilliant Blue-R250 (CBB) 10% methanol and 0.5% acetic acid, and then destained in a solution containing 10% methanol and 0.5% acetic acid. The other one gel was used for western blot analysis with mRib72-pAb.