Anti akt antibody

Animals were anesthetized with 1% pentobarbital, followed by an intraperitoneal (i.p.) injection of IS20 (0.5 mg/kg). The mice were sacrificed 10 or 20 minutes after the injection and tissues were harvested for protein extraction [26 (link)]. Tissue samples were homogenized in a lysis buffer consisting of 50 mM Tris-HCl pH 6.8, 1 mM EDTA pH 8.0, 1% NP-40, 1 mM Na₃VO₄, 0.1% SDS, 100 mM NaCl, and phosphatase and protease inhibitors. Homogenized samples or lysed CHO cell samples were incubated for 1 h at 4°C, with shaking, and were then centrifuged at 13,000 rpm and 4°C to obtain protein extracts. Protein concentration was determined with the BCA assay (Thermo Scientific), according to the manufacturer’s instructions. About 50 μg of protein for each sample was subjected to SDS-PAGE in a 12% polyacrylamide gel. The resulting bands were transferred to a PVDF membrane, which was incubated with anti-phospho-Akt serine 473 or anti-phospho-ERK antibodies (both at a dilution of 1:1000; anti-Akt antibody supplied by Santa Cruz and anti-ERK by Cell Signaling). Samples were treated with peroxidase-conjugated secondary antibody (Santa-Cruz; 1:5000 dilution), and immunoreactivity was detected with an ECL chemiluminescence detection kit, according to the manufacturer’s instruction (Amersham Pharmacia). The intensity of the resulting bands was determined with Image J software.

For immunoprecipitation, whole cell lysate were pre-cleared with Protein A/G plus (Santa Cruz Biotechnology Inc.) for 30 min at 4°C. Beads were pelleted at 1000 × g for 30 s and pre-cleared supernatants were incubated with 15 μg of primary antibody-agarose conjugates at 4°C overnight on a rotator. When agarose or a gel conjugate was unavailable, lysates were incubated with anti-Akt antibody (Santa Cruz Biotechnology Inc.) for 2 hrs at 4°C and then overnight along with Protein A/G plus beads to collect the immune complexes. Beads were collected by centrifugation, washed several times with RIPA buffer, one wash with PBS, and resuspended in SDS-PAGE sample loading buffer. Immune complexes and 80 μg of proteins were resolved by SDS-PAGE. Proteins were blotted onto PVDF Hybond membranes, which were then incubated overnight at 4°C with (mouse) anti-Akt antibody (mouse) anti-pAkt (mouse) anti-Ac-lysine (Santa Cruz Biotechnology Inc.). After washing, membranes were incubated with peroxidase-conjugated secondary antibodies for 1 hr. Immunolabelled bands were detected with a SuperSignal West Dura (Pierce).