Hybond lfp polyvinylidene difluoride pvdf membranes

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS/PAGE) was performed using 10% polyacrylamide gels. Proteins were transferred to Hybond^®-LFP polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Chicago, IL, USA) using the Trans-Blot^® TurboTM transfer system (Bio-Rad, Hercules, CA, USA) at 200 mA/membrane for 30 min. The PVDF membranes were blocked with 5% dry, non-fat milk (wt/vol) in phosphate buffered saline (PBS; 8.07 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 137 mM NaCl, 0.27 mM KCl, pH 7.2) containing 0.05% Tween-20 (PBS-T) for 1 h at 20 °C before immunoblotting with the indicated antibody in blocking solution overnight at 4 °C. The PVDF membranes were washed with PBS-T three times (5 min each) before incubation with HRP-conjugated rabbit anti-mouse IgG (1/10,000) or HRP-conjugated goat anti-rabbit IgG (1/30,000) in blocking solution at 20 °C for 2 h. After washing the PVDF membranes with PBS-T three times (5 min each), the immunoreactive bands were developed using a chemiluminescent detection kit (Thermo Fisher Scientific) and detected with an Amersham Imager 600 (GE Healthcare Europe, Barcelona, Spain).

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS/PAGE) was performed using 7% polyacrylamide gels. Proteins were transferred to Hybond-LFP polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Chicago, IL, USA) using the Trans-Blot Turbo™ transfer system (Bio-Rad, Hercules, CA, USA) at 200 mA/membrane for 30 min. PVDF membranes were blocked with 5% (wt/vol) dry non-fat milk in phosphate-buffered saline (PBS; 8.07 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 137 mM NaCl, 0.27 mM KCl, pH 7.2) containing 0.05% Tween-20 (PBS-T) during 1 h at 20 °C before being immunoblotted with the indicated antibody in blocking solution overnight at 4 °C. PVDF membranes were washed with PBS-T three times (5 min each) before incubation with either a HRP-conjugated rabbit anti-mouse IgG (1/10,000) or HRP-conjugated goat anti-rabbit IgG (1/30,000) in blocking solution at 20 °C during 2 h. After washing the PVDF membranes with PBS-T three times (5 min each), the immunoreactive bands were developed using a chemiluminescent detection kit (Thermo Fisher Scientific) and detected with an Amersham Imager 600 (GE Healthcare Europe, Barcelona, Spain).

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS/PAGE) was performed using 10% polyacrylamide gels. Proteins were transferred to Hybond^®-LFP polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Chicago, IL, USA) using the Trans-Blot^®TurboTM transfer system (Bio-Rad, Hercules, CA, USA) at 200 mA/membrane for 30 min. PVDF membranes were blocked with 5% (wt/vol) dry non-fat milk in phosphate-buffered saline (PBS; 8.07 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 137 mM NaCl, 0.27 mM KCl, pH 7.2) containing 0.05% Tween-20 (PBS-T) during 1h at 20 °C before being immunoblotted using mouse anti-A_2AR (0.5 µg/mL) and rabbit anti-α-actinin (0.5 µg/mL) antibodies in blocking solution overnight at 4 °C. PVDF membranes were washed with PBS-T three times (5 min each) before incubation with either a HRP-conjugated rabbit anti-mouse IgG (1/10,000) or HRP-conjugated goat anti-rabbit IgG (1/30,000) in blocking solution at 20 °C during 2 h. After washing the PVDF membranes with PBS-T three times (5 min each), the immunoreactive bands were developed using a chemiluminescent detection kit (Thermo Fisher Scientific) and detected with an Amersham Imager 600 (GE Healthcare Europe, Barcelona, Spain).