7 cm ipg strip

Aliquots of the IPVL extracts (100 µg) in a final volume of 125 µL of rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS wt/vol, 40 mM DTT, and 2% pharmalyte 3-10 NL), were applied to 7 cm IPG strip (pH range 3 to 10 NL; GE Healthcare). Then, IEF and two-dimensional electrophoresis were conducted as described in our previous study [115 (link)]. After 2DE, hen IPVL proteins were transferred to a nitrocellulose (NC) membrane (0.2 µm, Sigma-Aldrich) in a Mini Trans-Biol Cell (Bio-Rad, Hercules, CA, USA), according to Słowińska et al. [116 (link)]. The NC membranes were incubated overnight at 4 °C with anti-ubiquitin antibodies (U5379, Sigma-Aldrich) diluted with TBS-T (0.05 M Tris-HCl, 0.15 M NaCl, and 0.1% Tween 20; pH 7.6) at a ratio of 1:100. Blots were scanned with a VersaDoc MP 4000 system (Bio-Rad). The proteins of interest were excised manually, according to the Western signal, and prepared for mass spectrometry identification according to the protocol developed by Luque-Garcia et al. [117 (link)]. Antibodies were removed by three washes of the protein spots with 20 mM sodium bicarbonate buffer (pH 7.4), followed by washing with 100 mM glycine (pH 2.4). On-membrane digestion and sample preparation for mass spectrometry were performed according to our previous study [16 (link)].

A preparative 2D-mini gel (7 cm IPG strip, GE Healthcare, Chicago, IL, USA) stained Coomassie (Brilliant Blue G, Sigma Aldrich, Darmstadt, Germany), containing an equal amount of each non-labeled individual sample mixed with 10 µg of IS labeled sample, in a total of 75 µg of protein, was performed for further spot cut-off for mass spectrometry (MS) protein identification. The introduction of some labeled sample into preparative gel facilitates gel match with analytical gels for spot location and picking for MS analysis. The protein spot of interest was cut off from the gel and trypsin digested, as we previously described [16 (link),17 (link)]. Tryptic peptides, suspended in 50% (v/v) ACN and 0.1% (v/v) trifluoroacetic acid (TFA, for HPLC, ≥99.0%, Sigma Aldrich, Darmstadt, Germany) and submitted to sonication in an ultrasonic bath during 15 min, were directly applied on a 100-well matrix-assisted laser desorption/ionization (MALDI) plate with 5 mg/mL α-cyano-4-nydroxycinnamic acid (α-CHCA, 1:1) prepared in 0.1% TFA/60% ACN (Merck, Darmstadt, Germany) (v/v) and allowed to co-crystallize at RT. Peptides were analysed on an Applied Biosystems SCIEX 4800 Plus MALDI Proteomics Analyser with time-of-flight/time-of-flight (TOF/TOF) ion optics exactly as described [18 (link)]. The identified proteins are shown in Table S2 (Supplementary Materials).

All chemicals and reagents were from Sigma-Aldrich. Dried proteins (100 μg) were solubilized in 150 μl of 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 3–5.6) (GE Healthcare), and bromophenol blue]. After vigorous shaking, proteins were loaded onto 7-cm IPG strip (pH 3–5.6, GE Healthcare) by overnight passive rehydration at room temperature. The first-dimensional isoelectric focusing (IEF) was carried out on a Protean IEF Cell (Bio-Rad) using the following program: 300 V for 30 min (rapid voltage ramping), 1000 V for 1 h (rapid voltage ramping), 5000 V for 2 h (rapid voltage ramping), and 500 V for 3 h (rapid voltage ramping), with a current limit at 50 μA/gel. When IEF was complete (10,000 VH final), strips were 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] containing 1.5% (w/v) DTT then 2% (w/v) iodoacetamide. The second dimension separation was performed using 16.8% SDS–PAGE in Mini PROTEAN Tetra Cell (Bio-Rad).