All lanes : Anti-Ret (phospho Y1062) antibody (ab123544) at 1 µg/mlLane 1 : K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell LysateLane 2 : K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate with Immunising peptide at 1 µg/mlLane 3 : K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate with Control peptide at 1 µg/mlLysates/proteins at 25 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 Ret (phospho Y1062) staining in human prostate carcinoma 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 ab123544, 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.
ab123544 was tested using an Indirect ELISA approach. The wells were coated with peptide (1µg/ml at 100µl/well) overnight at 4°C, followed by a 5% BSA blocking step for 1 hour at room temperature. The primary Ab was then added at a dilution range of 1- 0.00025µg/ml (100µl/well) for 1hr at room temperature. A HRP-conjugated anti-rabbit IgG (heavy and light chain) was used as a secondary antibody at 1:20,000 dilution for 1hr at room temperature.