Protean i12 ief system

2D electrophoresis was conducted according to the procedure described by Szewczyk et al. (2015 (link)) with some modifications. The protein samples (600 μg) were loaded in 17-cm nonlinear IPG strips, pH 3–11 (Bio-Rad, USA). Isoelectric focusing (IEF) was performed using the Protean i12 IEF system (Bio-Rad, Germany) as follows: 50 V for 4 h, gradient to 6000 V for 5 h, and 50,000 Vh at 6000 V. The focused IPG strips were subjected to an additional reduction and alkylation treatment before the second dimension SDS-PAGE. The proteins were then separated in a 12% running gel by using a Protean XL cell (Bio-Rad, Germany) with the molecular mass marker of 6500–200,000 Da (Sigma-Aldrich, Germany) and stained using Coomassie blue. The gel analyses were performed using the Image Master 2D Platinum 7 software (GE Healthcare, Germany).

Immediately before 2D-PAGE analysis, 50 µg of protein from the samples was dissolved in 125 µL of buffer containing 7 M urea, 2 M thiourea, 4% CHAPS and 30 mM TRIS (pH 8.8).
All samples contained 50 µg of protein each were placed on 11 cm IPG strips with a linear pH range of 3–10. The strips were covered with mineral oil and active rehydration was carried out at 50 V for 12 h at 15 °C. Focusing began at 500 V for 30 min, continued at 1000 V for 30 min and was completed at 5000 V until a total value of 4.5 kVh was attained. Focusing was performed in a PROTEAN^® i12™ IEF System (Bio-RAD, USA).
After focusing the strips were equilibrated in reducing buffer (100 mM dithiothreitol; DTT, 6 M urea, 30% v/v glycerol, 2% SDS, pH 8.8) twice for 10 min each time, and then in alkylating buffer (150 mM IAA, 6 M urea, 30% v/v glycerol, 2% SDS) twice for 10 min each time at room temp. Second-dimension separation was carried out in a PROTEAN^® II xi Cell (Bio-RAD, Warsaw) in 12.5% polyacrylamide gel at a constant current of 25 mA/gel for 6 h at 15 °C (O’Farrell 1975 (link)).
Statistical analysis of the 2D electropherograms obtained was performed in ImageMaster 2D Platinum 7.0 software (USA).

The dried culture supernatant was dissolved in sample buffer (8 M urea, 2%CHAPS, 50 mM dithiothreitol [DTT], 0.2% Bio‐Lyte 3/10 carrier ampholyte, 0.001%BPB, #1632108 Bio‐Rad Laboratories, Inc., Hercules, CA, USA) and adjusted to 0.4 mg/ml. A total of 125 μl (50 μg) of the prepared samples was applied to immobilized pH gradient (IPG) strips (ReadyStrip, pH 3‐10 non‐linear, # 1632002, Bio‐Rad Laboratories, Inc.), and isoelectric focusing (IEF) was performed using the PROTEAN® i12 IEF System (Bio‐Rad Laboratories, Inc.) according to the manufacturer's instructions. Later, the IPG strips were reduced with DTT, carbamidemethylated with iodoacetamide, applied to precast gels (Any kD Mini‐PROTEAN TGX Precast Gel # 4569031, Bio‐Rad Laboratories, Inc.), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) was performed. The electrophoresed gels were stained with Flamingo Fluorescent Gel Stain (# 1610491, Bio‐Rad Laboratories, Inc.) or Coomassie Brilliant Blue (CBB) (Expedeon, Cambridge, UK). Image analyses of the stained gels were performed using a Gel Imaging system (Gel Doc EZ system, Bio‐Rad Laboratories, Inc.) and PDQuest 2‐D analysis software (Bio‐Rad Laboratories, Inc.).

A volume equivalent corresponding to 100 µg myelin protein was mixed with the same volume of rehydration buffer I (7 M urea, 2 M thiourea, 2% (w/v) ASB-14, 40 mM DTT, 1% (v/v) Servalyte ampholytes pH 3–10 (Serva)) and solubilized by short sonication and gentle shaking. The sample was filled up to 130 µL with rehydration buffer II (7 M urea, 2 M thiourea, 2% (w/v) ASB-14, 20 mM DTT, 0.5% (v/v) Servalyte ampholytes pH 3–10 (Serva)), solubilized as above, centrifuged (2 min 16,000× g), and the supernatant subjected to IEF in immobilized pH gradients (IPG BlueStrips, 7 cm, pH 3–10 or 3–12, Serva) in a Protean i12 IEF System (BioRad, Munich, Germany). During active rehydration at 50 V, 20 °C for ~16 h, current was limited to 70 µA. After 6 h active rehydration, paper wigs moisturized with distilled water were placed at both electrode ends underneath the IPG strips. For isoelectric focusing a step-gradient protocol was set according to the manufacturer’s instructions (1: hold at 150 V for 1 h; 2: hold at 300 V for 1 h; 3: ramp to 1000 V in 1 h; 4: ramp to 3000 V in 2 h; 5: hold at 3000 V for 2 h) at 20 °C.

All chemicals and reagents were from Sigma-Aldrich, unless specified. For two-dimensional electrophoresis (2D-E), 5 μg of dried proteins were solubilized in 150 μl of the rehydration buffer (8 M Urea, 2 M Thiourea, 2% (w/v) CHAPS, 10 mM dithiothreitol (DTT), 1.2% (v/v) Immobilized pH Gradient (IPG) buffer (pH 4-7) (GE Healthcare) and bromophenol blue). After vigorous shaking, proteins were loaded onto a 7-cm IPG strip (pH 4–7, Bio-Rad) by overnight passive rehydration at room temperature. The first-dimensional isoelectric focusing (IEF) was carried out on a PROTEAN® i12™ IEF system (Bio-Rad) using the following program: 250 V for 30 min (rapid voltage ramping), 1,000 V for 1 h (gradual ramping), 5,000 V for 2 h (gradual ramping) and held at 5,000 V (rapid ramping voltage) until complete IEF (10,000 VH final), with a current limit at 50 μA/gel. Strips were then incubated twice for 15 min in the equilibration buffer (375 mM Tris-HCl pH 8.8, 6 M urea, 2% (w/v) SDS and 30% (v/v) glycerol) complemented with 1.5% (w/v) DTT, followed by 15 min in the equilibration buffer complemented with 2% (w/v) iodoacetamide. The second-dimension separation, as well as mono-dimensional electrophoresis, were performed using 16.8% SDS–PAGE in Mini PROTEAN® Tetra Cell (Bio-Rad) as already described (19 (link)).

For 2-DE, dried proteins were resuspended in 10 μL water, then in 120 μL of rehydration buffer as already described. Immobiline Dry Strips (IPG strips: pH 3–5.6, 7 cm, GE Healthcare or pH 4–7, 7 cm, Bio-Rad) were submitted to passive rehydration with samples during 16 h at room temperature (RT). First dimension (isoelectrofocalisation) was performed on PROTEAN i12™ IEF system (BioRad), with the standard program “7-cm Gradual S-1” (250 V rapid for 30 min, 1,000 V gradual for 1 h, 5,000 V gradual for 2 h and a hold of 5,000 V), with a current limited to 50 μA/gel. When IEF was complete (9,000 VH final), IPG strips were incubated successively for 15 min in the equilibration buffer (375 mM Tris-HCl pH 8.8, 6 M Urea, 2% (w/v) SDS and 30% (v/v) glycerol) containing first 1.5% (w/v) DTT then 2% (w/v) iodoacetamide. The second dimension was performed using 16.8% acrylamide SDS-PAGE in Mini-PROTEAN^® Tetra cell (Bio-Rad) with Precision Plus Protein Standard Dual Color (Bio-Rad) as molecular mass markers.

Labeled and precipitated proteins were resuspended in UTC buffer (8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1 g AG 501-X8 Resin) containing 1% (v/v) ampholyte and 65 mM DTT. Samples were isoelectrically focused on 7 cm immobilized pH gradient (IPG) 3–10 pH strips (BioRad–ReadyStrip^TM IPG Strips) using BioRad PROTEAN i12 IEF system with the following focusing conditions: 12 h passive rehydration; 250 V, 15 min, rapid ramp; 4000 V, 1 h, slow ramp; 4000 V, 30000 Vhr, rapid ramp; 500 V hold. After focusing, IPG strips were equilibrated in IEF Equilibration buffer [6 M urea, 5% SDS (w/v), 30% glycerol (v/v)] containing 1 % (w/v) DTT, then in IEF Equilibration buffer containing 2.5% iodoacetamide (w/v). The second dimension electrophoresis was run on a 15% SDS gel. Gels were imaged using a Typhoon 9400 Imager (GE Healthcare) using excitation and emission wavelengths of 532/580 nm. Images were quantified using ImageJ 1.48V by multiplication of the fluorescence intensity and the area of each of the spots (n = 3).