Clarity ecl detection kit

Cell protein samples were obtained via cell lysis in a sample buffer (62.5 mM Tris-HCl pH 6.8, 2.5% SDS, 0.002% Bromophenol Blue, 5% β-mercaptoethanol, 10% glycerol). Proteins were divided by the SDS-PAGE method. Afterward, proteins were transferred from polyacrylamide gel to the PVDF membrane by Western blotting. TBS containing 0.1% Tween-20 and 5% BSA (PanEco, Moscow, Russia) was used to prevent non-specific binding. The next step was overnight staining of the membrane with protein-specific antibodies to phosphorylated T308 Akt [Anti p-Akt (Thr308) (244F9) Rabbit mAb #4056; Cell Signaling Technology Inc., Danvers, MA, United States] and Vinculin (Anti Vinculin Rabbit antibody V4139; Sigma-Aldrich). Unbound antibodies were then washed away, and the rest were incubated with antibodies for total rabbit immunoglobulins conjugated with peroxidase (P-GAR Iss; IMTEK, Moscow, Russia) for 1 h. Amplified chemiluminescence was used as a visualization method with a Clarity ECL detection kit (Bio-Rad). Image registration was carried out using the ChemiDoc Touch gel documenting system (Bio-Rad). Image analysis and volume measurements were performed using the Image Lab Software (Bio-Rad). Total Akt staining volume readings were normalized to the respective vinculin level, and then volume readings for p-Akt were compared with respective normalized Akt.

Treated cells were lysed in sodium dodecyl sulfate (SDS) lysis buffer [0.5 M Tris-HCl pH 6.8, 2% SDS (w/v) and 10% glycerol (v/v)] and heated at 95 °C for 10 minutes. The cell lysate was then centrifuged at 10,000 x g for 15 min at 4 °C, after which the supernatant was collected and the protein was determined using a BCA protein assay kit (Pierce Biotechnology, Illinois, USA). Cell lysate (total protein of 25–35 µg) from each treated condition was resolved on SDS polyacrylamide gels under reducing conditions and electrotransferred onto a PVDF membrane (Pall Corporation, New York, USA). After blocking with 5% non-fat milk in Tris buffer saline (TBS) containing 0.05% Tween-20 (TBS-T), the membrane was incubated with primary antibodies for 1 hour at room temperature. Bound antibodies were then detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgGs for 1 hour. After extensive washing, immunoreactive protein was visualized with a chemiluminescence-based procedure using the Clarity ECL detection kit (Biorad Laboratories, California, USA) and x-ray film (Kodak, New York, USA)