Tris hcl gradient sds page gels

For western blot analysis of A6 cells transfected with siRNAs, cells were lysed in lysis buffer (50mM Tris-HCl pH7.4, 150 mM NaCl, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM DTT and protease inhibitor cocktail) 48hours after the second transfection. The lysates were sonicated on ice, centrifuged at 20,000 × g for 15 min, and then Laemmli buffer was added to the supernatant. The samples were heated at 98 °C for 5 min, and then subjected to SDS-PAGE with precast 4–15% Tris-HCl gradient SDS-PAGE gels (Bio-Rad). Proteins from the SDS-PAGE gels were then transferred onto polyvinylidene fluoride membranes (Bio-Rad). Western blotting was performed with a mouse monoclonal anti-Talin antibody (clone 8d4, Sigma-Aldrich, T3287) or a mouse monoclonal anti-ß-actin antibody (clone AC-74, Sigma-Aldrich, A2228) as the primary antibody and HRP-conjugated anti-mouse IgG antibody (eBioscience) as the secondary antibody.

For western blot analysis of A6 cells transfected with siRNAs, cells were lysed in lysis buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM DTT and protease inhibitor cocktail) 48 h after the second transfection. The lysates were sonicated on ice, centrifuged at 20,000 × g for 15 min, and then Laemmli buffer was added to the supernatant. The samples were heated at 98 °C for 5 min, and then subjected to SDS-PAGE with precast 4-15% Tris-HCl gradient SDS-PAGE gels (Bio-Rad). Proteins from the SDS-PAGE gels were then transferred onto polyvinylidene fluoride membranes (Bio-Rad). Western blotting was performed with a mouse monoclonal anti-Talin antibody (clone 8d4, Sigma-Aldrich, T3287) at a dilution of 1:100 or a mouse monoclonal anti-ß-actin antibody (clone AC-74, Sigma-Aldrich, A2228) at a dilution of 1:1000 as the primary antibody and HRP-conjugated anti-mouse IgG antibody (eBioscience) at a dilution of 1:1000 as the secondary antibody.