Immobilon fl polyvinylidene fluoride pvdf membranes

The cardiac tissue protein was extracted, and the protein concentration was assessed, as previously described (Ye et al., 2018 (link)). Protein was separated by electrophoresis using Laemmli sodium dodecyl sulfate (SDS)-polyacrylamide gels and then transferred to Immobilon-FL polyvinylidene fluoride (PVDF) membranes (Millipore, United States). Subsequently, after being blocked with 5% non-fat milk for 1 h, the membranes were incubated at 4°C overnight with the following primary antibodies: ChemR23 (Santa Cruz Biotechnology, United States), Bax [Cell Signaling Technology (CST), United States], Bcl-2 (Abcam, United States), c-caspase 3 (CST, United States), phosphorylated/total-p65 (p/T-P65; CST, United States), phosphorylated/total-extracellular signal-regulated kinase (p/T-ERK; CST, United States), phosphorylated/total-c-Jun N-terminal kinase (p/T-JNK; CST, United States), phosphorylated/total-p38 mitogen-activated protein kinase (p/T-P38 MAPK; CST, United States), and GAPDH (CST, United States). Then, the membranes were treated with a second antibody at room temperature for 1 h. Finally, antibody binding was detected with a two-color infrared imaging system (Odyssey, LI-COR Biosciences, Lincoln, United Kingdom). The protein expression intensity was normalized to that of GAPDH.

Immobilon-FL polyvinylidene fluoride (PVDF) membranes (Millipore) were pre-activated by immersing in 100% methanol for 30 sec and then washing in dH₂O for 2 min. The membrane, along with two 0.5 mm filter pads, was soaked in PBS for 10 min in order to equilibrate in the buffer. A dry filter pad was placed on top of a bench, followed by the soaked filter pad and finally the PVDF membrane. Various amounts (1 µg, 0.5 µg, 0.25 µg, 0.125 µg, 0.0625 µg, 0.03 µg, 0.015 µg, 0.0075 µg) of MBP-fusion proteins were then adsorbed onto their respective grids in the membrane and left to dry on the bench, at RT, for 2 h. The blot was then blocked in 15 mL of 5% non-fat dry milk in PBS for 1 h in RT, followed by three washes in PBS for 5 min. Approximately 10 nM each of A10 scFv, A10 scFv-Fc and anti-α1 antibody (Abcam) was added to the appropriate strips and incubated overnight at 4°C with shaking. The binding of scFv and scFv-Fc versions of A10 was detected with α-Flag-800CW (1∶15,000; Rockland Immunochemicals, Gilbertsville, PA) and anti-α1 with goat, anti-rabbit HRP (1∶5,000; Pierce) for 45 min at RT. The membrane was washed three times and imaged with the Li-COR imager dual channel (800 nm and chemiluminescent) device (Odyssey Li-COR Biosciences; Lincoln, NE).