WB
Western blot analysis of Phospho-MEK1 (S298) on different lysates with Rabbit anti-Phospho-MEK1 (S298) antibody at 1/5,000 dilution.
Lane 1: HeLa,
Lane 2: A431,
Lane 3: NIH/3T3,
Lane 4: Mouse brain,
Lane 5: Rat brain,
Lane 6: Mouse heart,
Lane 7: Rat heart,
Lysates/proteins at 20 µg/Lane.
Exposure time: 25 seconds;
Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody:1/5,000 in primary antibody dilution buffer, overnight at 4 ℃
Secondary antibody: Goat anti-Rabbit IgG-HRP, 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature.
IHC
Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue using anti-Phospho-MEK1 (S298) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (1/50) for 30 minutes 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.
ICC/IF
ICC staining of Phospho-MEK1 (S298) in SW480 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).