Immobiline

The protein profile quality of shoot apexes was evaluated using 20 μg of protein submitted to SDS-PAGE gels (8 × 10 cm, acrylamide 12,5%) in vertical electrophoresis system (Omniphor).
To the 2D analyses, 500 μg of proteins were applied in immobilized pH gradient (IPG) gel strips of 13 cm with pH range of 3–10 NL (Amersham Biosciences, Immobiline™ Dry-Strip). The isoelectric focusing was carried out in the Ettan IPGphor 3 (GE Healthcare) system, controlled by Ettan IPGphor 3 software. Electrofocusing conditions: rehydration time – 12 h at 20 °C; Running - 500Vh for 1 h, 1000Vh for 1:04 h, 8000Vh for 2:30 h and 8000Vh for 40 min. The strips were reduced using equilibrium buffer (urea 6 mol L^− 1, Tris-HCl pH 8.8 75 mmol L^− 1, glycerol 30%, SDS 2%, bromophenol blue 0.002%) with DTT 10 mg mL^− 1 for 15 min, and alkylated using equilibrium buffer with iodoacetamide 25 mg mL^− 1 for 15 min. Finally, strips were equilibrated with running buffer (Tris 0.25 mol L^− 1, glycine 1.92 mol L^− 1, SDS 1%, pH 8.5) for 15 min. The second dimension was carried out in polyacrylamide gels 12.5% (triplicates) and the electrophoresis running were performed in the HOEFER SE 600 Ruby (GE Healthcare) vertical electrophoresis system under the following parameters: 15cmA/gel for 15 min, 40 mA/gel for 30 min and 50 mA/gel for 3 h, or until complete migration of sample trough the gel. After fixation and coloration with colloidal Comassie Brilliant Blue (CBB) G-250, gels were decolorized with distillated water. The digitalization process was made using ImageScanner III (GE Healthcare), the images were analyzed, and the spot detection was made by matching the gels triplicates in silico using Image Master 2D Platinum software (GE Healthcare).

To extract proteins from the spermatozoa, 50 × 10⁶ spermatozoa were incubated in rehydration buffer containing 7 M urea (Sigma, St Louis, MO, USA), 2 M thiourea (Sigma, St Louis, MO, USA), 4% (w/v) CHAPS (USB, Cleveland, OH, USA), 0.05% (v/v) Triton X-100 (Sigma, St Louis, MO, USA), 1% (w/v) octyl β-D-glucopyranoside, 24 μM PMSF (Sigma, St Louis, MO, USA), 1% (w/v) DTT (Sigma, St Louis, MO, USA), 0.5% (v/v) IPG Buffer, and 0.002% (w/v) bromophenol blue at 4°C for 1 h. Then, 250 μg of solubilized protein from the sperm cells in 450 μL of rehydration buffer was placed in a rehydration tray with 24 cm-long NL Immobiline DryStrips (pH 3–11; Amersham, Piscataway, NJ, USA) for 12 h at 4°C. First dimension electrophoresis was performed using an IPGphor IEF device and then the strips were focused at 100 V for 1 h, 200 V for 1 h, 500 V for 1 h, 1,000 V for 1 h, 5,000 V for 1.5 h, 10 8,000 V for 1.5 h, and 8,000-90,000 Vhr. After iso-electrofocusing, the strips were equilibrated a second After iso-electrofocusing, the strips were equilibrated with equilibration buffer A containing 6 M urea, 75 mM Tris–HCl (pH 8.8), 30% (v/v) glycerol, 2% (w/v) SDS, 0.002% (w/v) bromophenol blue, and 2% (w/v) DTT for 15 min at RT. The strips were equilibrated for a second time with equilibration buffer B (equilibration buffer A with 2.5% [w/v] iodoacetamide [Sigma] but without DTT for 15 min at RT. Next, 2-DE was carried out with 12.5% (w/v) SDS-PAGE gels with the strips at 100 V for 1 h and 500 V until the bromophenol blue front began to migrate off the gels. The gels were silver-stained for image analysis according to the manufacturer’s instructions (Amersham, Piscataway, NJ, USA). The gels were then scanned using a high-resolution GS-800 calibrated scanner (Bio-Rad, Hercules, CA, USA). Detected spots were matched and analyzed by comparing the gels from spermatozoa before- and after-capacitation using PDQuest 8.0 software (Bio-Rad, Hercules, CA, USA). The gel from before-capacitation spermatozoa was used as a control. Finally, the density of the spots was calculated and normalized as the ratio of the spot on the after-capacitation gel to that on the before-capacitation gel.

Two hundred μg of proteins, for each sample, were filled up to 400 μl in rehydration solution. Immobiline Dry-Strips (GE Health Care Europe; Uppsala, Sweden); 18 cm, linear gradient pH 3–10) were rehydrated overnight in the sample and then transferred to the Ettan IPGphor Cup Loading Manifold (GE Healthcare) for isoelectrofocusing (IEF). IEF was performed at 16°C and the proteins were focused for up to 70000Vh. The second dimension (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis; SDS-PAGE) was carried out by transferring the proteins to 12.5% polyacrylamide gel, running at 16 mA/gel and 10°C for about 16 h. The gels were stained with Ruthenium II tris (bathophenanthroline disulfonate) tetrasodium salt (SunaTech Inc.; Suzhou, P. R. China) (RuBP) as described by Aude-Garcia et al.[29 (link)]. Briefly, after the electrophoresis, the gels were fixed in 1% phosphoric acid (v/v) and 30% ethanol for 1h then were stained overnight with 1 mM RuBP in 1% phosphoric acid and 30% ethanol. After this time the gels were destained for 5 hours in 1% phosphoric acid and 30% ethanol and rinsed in water prior to acquisition by “ImageQuant LAS4010” (GE Health Care). The analysis of images was performed using Progenesis Same Spot (v4.1, Nonlinear Dynamics; Newcastle Upon Tyne, UK) software. This software generates 2DE analyses which are robust and accurate. Briefly, the gels were aligned to place all spots in exactly the same location, and afterwards, the spot detection produced a complete data set since all gels contain the same number of spots, each matched to its corresponding spot on all gels. After 2DE gel alignment and subsequent spot detection, the software calculated background corrected abundance, by determining the lowest intensity value of the image pixels outside the spot’s outlined, and subtracting it from the intensity value of every pixel inside the spot outline. Theses abundances were then normalized compared to a reference gel in order to obtain a normalized intensity value for each spot.
The 2DE experiments were performed in triplicate. The spot volume ratios between the two different conditions were calculated using the average spot normalized volume of the three biological replicates (http://www.nonlinear.com/). The software included statistical analysis calculations.

Reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise noted. Proteins were isolated from the aerial parts of WT and AtB-1 A. thaliana plants (1 g fresh weight) using a phenol extraction methanol/ammonium acetate precipitation method as described [19 (link)]. A protein from each extraction type was quantified using Bradford assay. For isoelectric focusing, dried protein pellets were dissolved in IPG buffer, containing 9.5 M urea with thiourea, 4% w/v CHAPS, 65 mM DTT, 2% Pharmalyte pH 3-10 (GE Healthcare, Uppsala, Sweden), and 0.01% w/ bromophenol blue. Protein probe diluted in IPG buffer was loaded to 18-cm Immobiline DryStrip pH 3–10 NL (GE Healthcare, Uppsala, Sweden) according to the manufacturer’s recommendations by passive rehydration for 12 h at 20 °C. IEF was performed in a Protean IEF Cell (Bio-Rad Laboratories Inc., Hercules, CA, USA) for 60,000 V-h as described [19 (link)]. For SDS-PAGE, 12% polyacrylamide gels with 4% stacking gels were run in a Protean II xi cell (Bio-Rad Laboratories Inc., Hercules, CA, USA). The gels were stained with Coomassie Brilliant Blue G-250. A set of three control and three experimental gels was used in the analysis.

Two-dimensional electrophoresis (2DE) was performed by the Immobiline polyacrylamide system. The first dimension was carried out on pH 3–10 nonlinear, 18 cm long, immobilized pH gradient strips (Cytiva, formerly GE Healthcare) using an Ettan™ IPGphor™ system (GE Healthcare, Uppsala, Sweden). Analytical runs consisted of an overnight rehydration of strips with 350 µL of lysis buffer and traces of bromophenol blue at room temperature and a loading of the samples at the cathodic end of the strips by cup-loading. Electrical conditions were as follows: 200 V for 8 h, from 200 to 3500 V in 2 h, 3500 V for 2 h, from 3500 to 5000 V in 2 h, 5000 V for 3 h, from 5000 to 8000 V in 1 h, 8000 V for 3 h, from 8000 to 10,000 V in 1 h, 10,000 V, for a total of 90,000 VhT (total Volts per hour) at 16 °C. Preparative runs were carried out by rehydration loading with 350 µL of the sample at 16 °C at 30 V overnight, then 100 µL of the sample were loaded at the cathodic end of the strips, with the following electrical conditions: 200 V for 8 h, from 200 to 3500 V in 2 h, 3500 V for 2 h, from 3500 to 5000 V in 2 h, 5000 V for 3 h, from 5000 to 8000 V in 1 h, 8000 V for 3 h, from 8000 to 10,000 V in 1 h, 10,000 V, for a total of 90,000 VhT (total Volts per hour) at 16 °C. Traces of bromophenol blue and carrier ampholyte at 0.2% for the analytical runs and at 2% for the preparative ones were added to the samples. After isoelectric focusing, an equilibration of the strips was performed as follows: first, incubation for 12 min in a solution composed of 6 M urea, 2% w/v SDS, 2% w/v DTE, 30% v/v glycerol and 0.5 M Tris–HCl pH 6.8 and a second incubation for 5 min—in a solution composed of 6 M urea, 2% w/v SDS, 2.5% w/v iodoacetamide, 30% v/v glycerol, 0.5 M Tris–HCl pH 6.8 and traces of bromophenol blue. Second dimension by SDS/PAGE was performed at 9 °C at 40 mA/gel constant current, using 9–16% SDS polyacrylamide linear gradient gels (18 cm × 20 cm × 1.5 mm in size). Then, ammoniacal silver staining was used for analytical gels, whereas MS-compatible silver staining was used for preparative gels. The resulting gels were scanned with Image Scanner III laser densitometer run by the LabScan 6.0 software (GE Healthcare). Image Master 2D Platinum 6.0 software (GE Healthcare, Uppsala, Sweden) was used to carry out 2D image analysis.