All lanes : Anti-IRAK (phospho T387) antibody (ab139739) at 1 µg/mlLane 1 : THP1 (Human acute monocytic leukemia cell line) Whole Cell LysateLane 2 : THP1 (Human acute monocytic leukemia cell line) Whole Cell Lysate with Human IRAK (phospho T387) peptide (ab174118) at 1 µg/mlLane 3 : THP1 (Human acute monocytic leukemia cell line) Whole Cell Lysate with Human IRAK peptide (ab174119) at 1 µg/mlLysates/proteins at 25 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
ab139739 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.