Immobilized ph gradient strips

The protein lysates for Mock and HCV transfected samples were precipitated using 10% TCA and pellets were washed with acetone. The protein pellets were resuspended in sample rehydration buffer (8 M urea, 2% CHAPS [wt/vol] 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 15 mM dithiothreitol [DTT], and 0.5% [vol/vol] IPG buffer [pH 3 to 10]). Immobilized pH gradient (IPG) strips (7 cm length) with pH range 3 to 10 were used for isoelectric focusing (Bio-Rad). For the first dimension, 500 μg of protein samples in 150 μl of rehydration solution was used to rehydrate the IPG strip. The IPG strips were passively rehydrated overnight, and then the proteins were focused for 10,000V. h at 20°C under mineral oil in PROTEAN IEF System (Bio-Rad). After focusing, the strips were incubated for 10 min in 2 ml of equilibrium buffer I (6 M urea, 30% glycerol [wt/vol], 2% SDS [wt/vol], and 1% DTT [wt/vol] in 50 mM Tris-HCl buffer [pH 8.8]), followed by equilibrium buffer II (6 M urea, 30% glycerol [wt/vol], 2% SDS [wt/vol], and 4% iodoacetamide [wt/vol] in 375 mM Tris-HCl buffer [pH8.8]). After the equilibration steps, the strips were transferred to a SDS-12% polyacrylamide gel electrophoresis (PAGE) gel for the second dimension electrophoresis. Protein spots were visualized by silver staining or transferred on Nitrocellulose membrane for western blotting.

The separation and illustration of the secretomes was carried out using 2-DE. The 2-DE was performed according to our established protocol [20 (link)]. The secretomes from cells treated with either ANG II, PDGF, or TGFβ1 and from control cells were prepared as described above and 150 µg proteins from each treatment and control were diluted in rehydration buffer (8 M urea, 1% (w/v) CHAPS, 0.2% ampholytes pH 5–8 for 11 cm IPG strips, 15 mM DTT, and a trace of bromophenol blue). The proteins were separated in the first dimension according to their isoelectric point on immobilized pH gradient (IPG) strips (pH 5–8, Bio-Rad). After the first-dimension separation and a subsequent equilibration step, the proteins were separated in a second dimension according to their molecular weight on a 12% SDS-PAGE. The 2-D gels were subjected to a fixation step (50% methanol and 12% acetic acid overnight) prior to staining with Flamingo fluorescent protein gel stain (Bio-Rad) for 5 h. The stained gels were then scanned at 50 µm resolution on a Fuji- FLA5100 (Fuji Photo, Kanagawa, Japan).