3. Experimental part
3.3 Proteins analysis by electrophoresis
• 2-Iodoacetamide IAA (CH2CONH2) (Sigma-Aldrich, USA)
• 50% glycerol (Bio-Rad, Germany)
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• Acetic acid (CH3COOH), (solution 10% in Milli Q H2O) (Carlo Erba, Italy)
• Agarose (Bio-Rad, Germany)
• Bromophenol blue (Bio-Rad, Germany)
• Carrier ampholytes (Bio-Rad, Germany)
• CHAPS (Bio-Rad, Germany)
• Coomassie Brilliant Blue R-250 (Bio-Rad, Germany)
• Criterion XT Precast Gel, 12% Bis-Tris, 12 wells (Bio-Rad, Germany)
• Criterion XT Precast Gel, 12% Bis-Tris, IPG+1 comb (Bio-Rad, Germany)
• Dithiothreitol DTT (HSCH2(CHOH)2CH2SH) (Sigma-Aldrich, USA)
• Methanol (CH3OH), HPLC grade, (Sigma-Aldrich, USA)
• Milli Q H2O obtained with Millipore Alpha Q system
• Mineral oil (Bio-Rad, Germany)
• Ready Strip IPG strips, 11 cm, pH: 4-11 (Bio-Rad, Germany)
• Sodium Dodecyl Sulphate SDS, in powder and in solution 20% (Sigma-Aldrich, USA)
• SDS-PAGE Molecular Weight Standards, Broad Range (Bio-Rad, Germany)
• Tris/HCl (50 mM solution in Milli Q H2O) (Sigma Aldrich, USA)
• Urea (NH2CONH2) (Sigma-Aldrich, USA)
• XT MES Running buffer 20X (Bio-Rad, Germany)
• XT sample buffer 4X (Bio-Rad, Germany)
• XT Reducing Agent 20X (Bio-Rad, Germany)
3.3.2 Instrumentation
• Amicon® Ultra-4 Centrifugal Filter Units Amicon Ultra 5 KDa device — 5,000 NMWL (Nominal Molecular Weight Limit) (Millipore, Italy)
• Centrifuge Universal 320 R (Hettich, Germany)
• Rotor 1620A (97 mm radius) (Hettich, Germany)
• Adapters 1451 (Hettich, Germany)
• Centrifuge 1-13 (Sigma-Aldrich, USA)
• Criterion Cell (Bio-Rad, Germany)
• GS-800 calibrated densitometer (Bio-Rad, Germany)
• Glass Syringe 50 μL (Hamilton, USA)
• Gyro-Rocker SSL3 (Stuart Scientific, UK)
• PDquest software (Bio-Rad, Germany)
• Power supply: Power Pac Universal (Bio-Rad, Germany)
• PROTEAN IEF Cell (Bio-Rad, Germany)
• PCR Express Thermal Cycler (Thermo Hybaid, UK )
3.3.3 Procedures for 1D gel electrophoresis i
i. . PrProotteiein n ssttaannddaarrdd pprreeppaarraattiionon
• 2 μL SDS-PAGE Molecular Weight Standards, Broad Range
• 28 μL of reducing sample buffer (prepared with 9.5 μL XT buffer 4X, 0.5 μL XT reduction buffer 20X and Milli Q H2O to final 30 μL volume)
• 5’ incubation at 95 °C
59 i
iii. . SSamamppllee pprreeppaarraattiionon
• XT sample buffer 4X, diluted 4 times
• XT Reduction buffer 20X, diluted 20 times
• 30 μg of proteins (or 1 mg, in case of semipreparative analysis), following protein sample quantification described in paragraph 3.2.3
• Milli Q H2O to final 25 μL volume (or 500 µL, in case of semi-preparative analysis)
• 5’ incubation at 95 °C
i
iiii.i. ElEleeccttrroopphhoorreettiicc rruunn
For each wells of the Criterion XT Precast gel, 25 μL of sample prepared (or 450 μL, in case of semi-preparative analysis) were loaded or 5 μL of protein standard. The running buffer used was XT MES Running buffer diluted 20 times, the voltage applied to the Criterion Cell was 150 V. The run lasted 60’. In the sample buffer there was a 1% of Bromophenol blue, which ran as indicator.
3.3.4 Procedures for 2D gel electrophoresis ii. . DDesesalalttiinngg
In this case the procedure required a desalting step.
PPrere--rriinnssiinngg: the ultra-filtration membranes in Amicon Ultra devices contained trace amounts of Polyethylene glycol (PEG). The device must be pre-rinsed with 5 washes of a CH3OH:H2O (1:1) solution.
P
Preresseerrvviingng: the devices were stocked in a CH3OH:H2O (5:95) solution, at 4 °C until use.
UUsese: the ultra-filtration device was loaded with 4 mL of the solution to be desalted and centrifuged. The sample was loaded and centrifuged at 6500 RCF, 4 °C for 45’, and then 3 washes with 4 mL of Milli Q H2O were performed. The filtered solution was discarded each time and the retentate (upper part of the device) was recovered after washes using an Eppendorf pipette P100. A major recovery was obtained adding 250 µL of Milli Q H2O, the membranes were washed using the pipette Pasteur and the recoveries were added to the suspension in an Eppendorf.
iiii. . FiFirrsstt ddiimmenenssiionon:: iissoeoelleeccttrrooffooccuussiingng
150 μg of proteins were re-dissolved in 185 μL of rehydration buffer, whose composition was: 8 M urea, 50 mM DTT, 4% CHAPS, 0.2% Carrier ampholytes, 0.0002% Bromophenol blue, Milli Q H2O to 5 mL. Re-dissolved samples were placed in the IEF tray, and the strips were settled with the active part in contact with the sample. Passive rehydration of the strips was allowed for 1 hour, after that 3 mL of mineral oil were added to cover completely the strips. The rehydration was performed then overnight.
The strips were then placed in the focusing tray: +/- strip direction follow +/- sense of the tray and 2.5 mL of mineral oil were added to the strips, in order to prevent evaporation.
The PROTEAN IEF CELL was programmed as follow:
Volt: 8000
Volt/hours: 29000
60 Ramp: rapid
Max µA/strip: 50
At the end of the focusing, strips were taken and exceeding mineral oil was discarder on filter paper.
iii. SStrtriipp eeqquuiililibbrraattiioonn
Strips were placed, with gel sided up, in a tray and proteins were reduced and alkylated by adding 4 mL of DTT Equilibration buffer for 10’ and, then, 4 mL of IAA Equilibration buffer for 10’, respectively to each strip. Equilibration base buffer was composed of: 6 M Urea, 2% SDS, 0.05 M Tris/HCl buffer pH 8.8, 20% glycerol, Milli Q H2O to 10 mL. 2% DTT Equilibration buffer was prepared immediately prior to use adding 200 mg of DTT. 2.5% IAA Equilibration buffer was prepared immediately prior to use by adding 250 mg of IAA.
i
iv.v. SSececoonndd ddiimmeennssiionon:: ggeell eelleeccttrroopphhoorreessiis s
Equilibrated strips were applied to the top of the Precast gels Criterion XT (12% Bis-Tris, IPG+1 comb). The strips were aligned so that the plastic back of the strip was against the back plate and the IPG strip was touching the top of the gel. “+” part of the strips was towards the well for the MW standards. MW standards were placed in the well (as described in paragraph 3.3.3). Molten agarose was added to fix the strips and, as it was set, the gels were covered with the XT MES Running buffer diluted 20 times.
The Criterion Cell was set for the run as described previously.
3.3.5 Gel staining
The staining solution was made up of 0.1% w/v of Coomassie brilliant blue R-250 dissolved in 10% CH3COOH, 40% CH3OH and Milli Q H2O to 1000 mL. Gels were placed in glass containers and were covered with the staining solution where they were allowed to soak for at least 1 hour. Any dye that was not bound to protein diffused out of the gel during the de-staining steps, when the gels were rinsed with a solution of 10% CH3COOH, 40% CH3OH, 50% Milli Q H2O, changed at least twice, to achieve the desired contrast.
3.4