Protean 2 xi

Next the soaked strips were subjected to isoelectric focusing (in the first dimension of electrophoresis) in the IEF-100 Hoefer apparatus (Hoefer IEF100, Hoefer, Inc., Holliston, MA, USA). The process conditions were as follows: 250 V/30 min; 10,000 V/3 h; 60 kV/h, with a current limit of 50 μA/strip. Before the second dimension, the focused strips were equilibrated sequentially in 1.4-dithiothreitol and iodoacetamide solutions. Each stage lasted 15 min. The equilibrated strips were subjected to molecular mass separation by vertical electrophoresis in 12.5% polyacrylamide gel placed in an electrophoretic chamber (PROTEAN^® II xi, Bio-Rad, Warsaw, Poland). Five repetitions of gels were performed. The current parameters were as follows: 600 V/30 mA/100 W. After separation, the gels obtained were silver stained with silver nitrate in the presence of formaldehyde and digitalised by scanning (Image Scanner III, GE Healthcare, Warsaw, Poland) [13 (link)].

The MHC profile consisting of the isoform composition of MHC I, MHC IIa, and MHC IIx was determined from muscle homogenate by using the SDS-PAGE gel electrophoresis and Bio-Rad Protean II Xi vertical slab gel system [25 ].

The protein solution was added at a ratio of 150 µg/IPG strip (Bio-rad). Lysis buffer 1 was added to make a total volume of 300 µL. Centrifugation was then performed at 30,000 g at 24°C for 30 min, and the supernatant was obtained for IPG strip rehydration. The rehydration was passive hydration, and the duration was 12–14 h at room temperature. After the rehydration, isoelectric focusing was carried out on a Protean IEF Cell (Bio-rad) with the following settings: S1 250 V 10 min, S2 500 V 30 min, S3 1000 V 1 h, S4 9000 V 5 h, S5 50000 VH (ramie sample)/55000 VH (other 5 plant samples), S6 500 V 1 h. After focusing, the strips were put into 5 mL of equilibrium buffer (6 M Urea, 2% SDS, 0.375 M, pH 8.8, Tris-HCl, 20% Glycerol); 0.05 g of DTT was added with gentle shaking on a shaker for 15 min in order to reach the first equilibrium; the strips were placed in the equilibrium buffer again; 0.255 g of IAA was added with gentle shaking for 15 min to reach the second equilibrium. A 12% polyacrylamide gel was used for the second dimension electrophoresis, the step was performed at 18°C in PROTEAN II XI (Bio-rad) with the following program: 10 mA, 1 h; 30 mA, 3.5 h. The SDS-PAGE two-dimensional electrophoresis of samples was undertaken a total of two times from two independent extractions.

IEF and SDS-PAGE were performed as described by Shen et al. [15 (link)]. The BioRad Protean IEF Cell was first used to rehydrate the 18 cm immobiline^TM dry strips (pH 4–7) (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) with a 300 µL rehydration buffer containing 200 µg of protein lysates made from mock and PEPE2-treated cells for 16 h at 20 °C. After rehydration, the proteins were concentrated at 20 °C under 50, 100, 200, 500, 1000, 5000, and 8000 V, respectively, with 81,434 voltage hours. Then, an equilibration buffer [2% (w/v) SDS (aMResco, Solon, OH, USA), cotaining 30% (v/v) glycerol (Kanto Chemical, Portland, OR, USA), and 6 M urea (aMResco, Solon, OH, USA)] with 2% (w/v) DTT (USB Corcorporation, Cleveland, OH, USA) was implemented to equilibrate the gel strips for 15 min. Afterwards, the DTT-equilibrated strips were incubated for 15 min with an equilibration buffer comprising 5% (w/v) iodoacetamide (aMResco, Solon, OH, USA). Then, the strips were put above a 12.5% (w/v) polyacrylamide gel sealed with 0.5% (w/v) agarose (aMResco, Solon, OH, USA) gel, and various proteins were resolved at 420 V using BioRad Protean IIxi until bromophenol blue run near the bottom.

The samples (labeled proteins) from the wild-type, mutant and mixed internal standard were dissolved in 250 μl of Rehydration-Buffer (7 M urea, 2 M thio-urea, 4% CHAPS with 0.5% DTT and 1.2% de-streaking solution) and 13 cm IPG strips with pH range 4–7 and 6–11 were rehydrated over night with the Rehydration-Buffer carrying the labelled proteome of the wild-type and the Δrrp8 mutant along with the internal standard. For the first dimension, the Ettan IPGphor-3 (GE Healthcare) was used to carry out isoelectric focusing (IEF). IEF was performed using the protocol mentioned in the GE handbook for respective pH range. Before subjecting the IPG strips to second dimension, an equilibration with Equilibration-Buffer (6 M urea, 20 mM Tris-HCl pH 8.8, 2% SDS, 20% glycerol) containing 1% DTT (freshly added) followed by Equilibration-Buffer containing 4% idoacetamide (added fresh) for 20 min each were carried out. After equilibration, IPG strips were blotted between two moist filter papers and then placed horizontally on to a polyacrylamide gel (12%) which was then sealed by 2% agarose containing bromophenol blue to perform the second dimension. The gels were run at 4 °C with constant current of 16 mA overnight using Biorad Protean II xi.

Nondenaturing PAGE was performed in a temperature-controlled electrophoresis cell (PROTEAN II xi, Bio-Rad) submerged in a cooling system. The gels (16%, 29:1 acrylamide/bisacrylamide) in 16 20-cm glass cassettes were electrophoresed for 22 h at 40 V and 7 °C in potassium phosphate buffer. The gels were stained with Stains-All (Sigma–Aldrich).