All lanes : Anti-mTOR (phospho S2448) antibody (ab84400) at 1 µg/mlLane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell LysateLane 2 : EGF-Stimulated A431 Whole Cell Lysate Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell LysateLysates/proteins at 10 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
All lanes : Anti-mTOR (phospho S2448) antibody (ab84400) at 1 µg/mlLane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell LysateLane 2 : EGF-Stimulated A431 Whole Cell LysateLane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell LysateLane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with Immunizing peptide at 1 µg/mlLane 5 : EGF-Stimulated A431 Whole Cell Lysate with Immunizing peptide at 1 µg/mlLane 6 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate with Immunizing peptide at 1 µg/mlLysates/proteins at 10 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
IHC image of mTOR staining in Human hippocampus formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab84400, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
ICC/IF image of ab84400 stained MCF-7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab84400 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.