Electrode wick

A protein sample (250 µg) was mixed with rehydration buffer (RB): 7M urea, 2M thiourea, 4% CHAPS, 0.05% triton X100, 0.5% ampholytes (IPG buffer 4–7 GE) and adjusted to the correct volume to rehydrate 18 cm strip (here, 320 µL). Strips were then placed acrylamide face down in the focusing tray equipped with platinum electrode embedded into the running tray (Protean IEF, Bio-Rad, Hercules, CA, USA) and passively re hydrated at 20°C without electricity for 16 h, and then actively rehydrated at 50 V during 9 h, as previously described [27 (link),35 (link),36 (link)]. During protein focalization, small Electrode wicks were placed between acrylamide and electrode. These paper wicks (Ref 1654071, Electrode wicks, Bio-Rad, Hercules, CA, USA) were, in advance, soaked with water in order to absorb salts and other contaminant species during active rehydration. The IPG strips were then focused according to the following program: 500 V for 1 h, a linear ramp to 1000 V for 1 h, a linear ramp to 10000 V for 33 KV-1 h, and finally 10000 V for 24 KV-1 h.

After rehydration, electrode wicks (Bio-Rad) were added to reduce artifacts. Focusing started with the “conditioning step” (2 h) which subdivides in 2 sub-steps: (a) linear voltage rise to 500 V, step-hold 30 min, (b) linear voltage rise to 2500 V, step-hold 1 h. After that, the “slow voltage ramping” (2.5 h): quadratic voltage rise to 8000 V and the “final focusing”: actual process of focusing (duration: 50.000 Vhrs) were performed. During the whole IEF the temperature was constantly kept at 20°C. After focusing the strips were stored at −80°C.

Acrylamide, bisacrylamide and Hybond™-P membrane were purchased from Amersham Bioscience/GE Healthcare (Little Chalfont, UK). IPG buffers, 18 cm ReadyStrip IPG strips (pH 5–8), 18 cm ReadyStrip IPG strips (pH 3.0-5.6), 18 cm ReadyStrip IPG strips (pH 5.3-6.5), and Electrode Wicks were from Bio-Rad Laboratories (Marnes la Coquette, France), and orthophosphoric acid, ammonium sulphate and absolute ethanol were from VWR (Strasbourg, France). All other chemicals were from Sigma (L’Isle-d’Abeau Chesnes, France). Sequence grade-modified trypsin was purchased from Promega (Charbonnières-les-bains, France), and Luminata™ Western Horseradish peroxidase (HRP) substrate and ReBlot Plus Strong antibody stripping solution were from Millipore (Molsheim, France). For immunoblotting, the monoclonal antibodies against VCL and FHL3 were from Sigma. The anti-mouse IgG-HRP, anti-rabbit IgG-HRP, anti-SRL and anti-β-actin (ACTB) were from Santa-Cruz biotechnology (Heidelberg, Germany). The polyclonal antibodies against GPD1, MYOZ1, ANXA1, ANXA5, GSTO1, HSPB5, HSPC3, ALDH2, NDUFS2, UQCRFS1, ENO3, HSPA1A and VCP were purchased from Euromedex (Genetex, Souffelweyersheim, France).