Ecl horseradish peroxidase linked anti mouse na931

For Western blotting of whole cell lysates, 5×10⁵ cells from overnight cultures were washed once with 10 mM Tris pH 7.4, resuspended in 500 μl 10 mM Tris, and precipitated with 10% TCA on ice for 30 min. Samples were then centrifuged at 12000 rpm (Eppendorf F-45-30-11 rotor) in an Eppendorf microfuge for 10 min at 4°C, washed once with ice-cold acetone an d resuspended in 100 μl 100°C SDS-PAGE sample buffer. Proteins were resolved with the Novex NuPAGE Gel system (4–20% Tris-Glycine gels, Invitrogen), and transferred to 0.2 μm PVDF membranes (Thermo Scientific). Blots were blocked with 5% dried milk or 3% BSA in 1X TBS-Tween (25 mM Tris, 3 mM KCl, 140 mM NaCl, 0.05% Tween 20, pH 7.4). The rabbit anti-Grl1p serum, the mouse mAb anti-GFP (BioLegend), the mouse mAb anti-c-myc (9E10, Sigma), the rabbit anti-FLAG (Sigma), and the mouse mAb anti-HA (HA.11, BioLegend) antibodies, were diluted 1:2000, 1:5000, 1:1000, 1:2000, 1:2000, respectively, in blocking solution. Proteins were visualized with either anti-rabbit IgG (whole molecule)-HPeroxidase (Sigma) or ECL Horseradish Peroxidase-linked anti-mouse (NA931) (GE Healthcare Life Sciences, Little Chalfont, UK) secondary antibody diluted 1:20000, and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).