Anti talin antibody clone 8d4

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.

Platelet lysates that contain 20 μg proteins were electrophoresed on 6% polyacrylamide gels for 45 minutes at 200 V. Proteins were transferred from gels onto 0.45μm polyvinylidene difluoride (PVDF) membranes (IPVH00010, Immobilon-P) for 1 hour at 100 V. Membranes were blocked with 5% w/v non-fat powdered milk in Tris-buffered saline-Tween (TBS-T; 137 mM NaCl, 20 mM Tris, 0.1% Tween-20, pH 7.6) for 1 hour at room temperature, incubated overnight with agitation at 4 °C with 1:10,000 anti-talin antibody (clone 8D4; T3287, Sigma) or 1:5000 anti-CD41 antibody (ab134131, Abcam) and washed three times with TBS-T. To detect the primary antibody, membranes were incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (7074 S, Cell Signaling Technology) or anti-mouse IgG (7076 S, Cell Signaling Technology), again washed three times with TBS-T. To visualise the blot, membranes were exposed to Super Signal Chemiluminescent Substrate (34077, Thermo Scientific) for 5 minutes. The blots were exposed to X-ray films (Amersham Hyperfilm ECL, GE Healthcare) and developed using an OptiMax X-ray film processor (Protec Medizintechnik).