Optimized 2D Gel Electrophoresis Workflow

Two hundred μg of proteins, for each sample, were filled up to 400 μl in rehydration solution. Immobiline Dry-Strips (GE Health Care Europe; Uppsala, Sweden); 18 cm, linear gradient pH 3–10) were rehydrated overnight in the sample and then transferred to the Ettan IPGphor Cup Loading Manifold (GE Healthcare) for isoelectrofocusing (IEF). IEF was performed at 16°C and the proteins were focused for up to 70000Vh. The second dimension (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis; SDS-PAGE) was carried out by transferring the proteins to 12.5% polyacrylamide gel, running at 16 mA/gel and 10°C for about 16 h. The gels were stained with Ruthenium II tris (bathophenanthroline disulfonate) tetrasodium salt (SunaTech Inc.; Suzhou, P. R. China) (RuBP) as described by Aude-Garcia et al.[29 (link)]. Briefly, after the electrophoresis, the gels were fixed in 1% phosphoric acid (v/v) and 30% ethanol for 1h then were stained overnight with 1 mM RuBP in 1% phosphoric acid and 30% ethanol. After this time the gels were destained for 5 hours in 1% phosphoric acid and 30% ethanol and rinsed in water prior to acquisition by “ImageQuant LAS4010” (GE Health Care). The analysis of images was performed using Progenesis Same Spot (v4.1, Nonlinear Dynamics; Newcastle Upon Tyne, UK) software. This software generates 2DE analyses which are robust and accurate. Briefly, the gels were aligned to place all spots in exactly the same location, and afterwards, the spot detection produced a complete data set since all gels contain the same number of spots, each matched to its corresponding spot on all gels. After 2DE gel alignment and subsequent spot detection, the software calculated background corrected abundance, by determining the lowest intensity value of the image pixels outside the spot’s outlined, and subtracting it from the intensity value of every pixel inside the spot outline. Theses abundances were then normalized compared to a reference gel in order to obtain a normalized intensity value for each spot.
The 2DE experiments were performed in triplicate. The spot volume ratios between the two different conditions were calculated using the average spot normalized volume of the three biological replicates (http://www.nonlinear.com/). The software included statistical analysis calculations.