Gapdh mca4739

Protein was isolated from liver tissue according to standard protocol in NP40-Lysis buffer.¹¹ (link) Concentrations were measured via Bradford assay (Bio-Rad, Hercules, USA) and normalized to 2 μg/μl. Then Laemmli buffer was added and protein samples were denatured at 96 °C for 10 min. The protein samples were separated via electrophoresis on precast 4%-12% polyacrylamide gel (Bio-Rad, Hercules, USA) while submerged in sodium dodecyl sulfate running buffer at 140 mV. Next, separated proteins were transferred to a nitrocellulose membrane via Trans-Blot Turbo Transfer System (Trans-Blot Turbo Transfer Pack 0.2 μm Nitrocellulose, Bio-Rad, Hercules, USA). A successful transfer of protein was confirmed by staining with Ponceau Red (Sigma, Steinheim, Germany). Then samples were incubated with 5% non-fat dry milk dissolved in tris-buffered saline tween (TBST) to block non-specific binding sites and treated with the primary antibody (Col1A1, 91144, Cell signaling, Danvers, USA; GAPDH, MCA4739, Bio-Rad, Hercules, USA) diluted in TBST overnight at 4 °C. On the next day, the membrane was washed with TBST and incubated with the horseradish peroxidase-conjugated secondary antibody (anti-rabbit for 1 h at room temperature). Next, the membrane was washed and incubated in ECL substrate (Pierce, Waltham, USA). The membrane was developed using LAS mini 4000 (Fuji, Tokyo, Japan).¹¹ (link)