WB
Western blot analysis of Phospho-mTOR (S2448) on different lysates with Mouse anti-Phospho-mTOR (S2448) antibody at 1/5,000 dilution.
Lane 1: HeLa cell lysate,
Lane 2: HeLa starved overnight then treated with 200nM PMA for 4 hours cell lysate,
Lysates/proteins at 20 µg/Lane.
Exposure time: 1 minute 30 seconds;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
IHC
Immunohistochemical analysis of paraffin-embedded human liver tissue with Mouse anti-Phospho-mTOR (S2448) antibody at 1/100 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody at 1/400 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.