Anti rabbit igg secondary antibody

Cultured fibroblasts were serum starved overnight prior to IGF-I treatment (15 or 20 min), cell lysates collected and subject to Western immunoblot analyses (19 (link)). Rabbit polyclonal IgG against phospho-AKT (dilution 1:1000) and rabbit monoclonal IgG against Akt (dilution 1:2000) were purchased from Cell Signaling Technologies (Beverly, MA, USA). The anti-rabbit IgG secondary antibody was purchased from Amersham-Pharmacia Biotech (Uppsala, Sweden).

Western blotting was performed using liver lysates as described previously [17 (link), 25 (link), 26 (link)]. In brief, proteins (50–80 μg) were separated on a 4–12% SDS-polyacrylamide gel with 2-(N-morpholine) ethane sulfonic acid or 3 (N-morpholino) propane sulfonic acid buffer purchased from Invitrogen (Carlsbad, CA, USA) at 200V. Gel was transferred on an Immuno-blot PVDF Membrane (Bio-Rad, Hercules, CA, USA) overnight at 4°C. Membranes were blocked in blocking solution (0.3% Tween 20 in Tris-buffered saline and 10% nonfat dry milk) for 1 h at room temperature then probed using rabbit polyclonal active caspase 2 (1:200), BAX (1:200), and BCL-2 (1:200) antibodies from Santa Cruz Biotechnology for 1 h at room temperature or overnight at 4°C with constant shaking. Following 3 X 10-min washes in TBS-T buffer, membranes were then incubated in anti-rabbit IgG secondary antibody (Amersham Biosciences, Piscataway, NJ, USA) at a 1:2000 dilution. For immunodetection, membranes were washed three times in TBS-T wash buffer, incubated with ECL solutions per manufacturer’s specifications (Amersham Biosciences), and exposed to Hyper film ECL. The membranes were stripped and re-probed with a rabbit polyclonal GAPDH (1:2000) from Millipore, Billerica, MA, for normalization of the loading. Band intensities were determined using Quantity One software from Bio-Rad.