WB
Western blot analysis of Phospho-c-Myc (T58) on different lysates with Rabbit anti-Phospho-c-Myc (T58) antibody at 1/1,000 dilution.
Lane 1: HCT 116 cell lysate,
Lane 2: HCT 116 treated with 25μM MG-132 for 4 hours cell lysate,
Lane 3: NIH/3T3 cell lysate,
Lane 4: NIH/3T3 treated with 10μM MG-132 for 8 hours cell lysate,
Lysates/proteins at 20 µg/Lane.
Exposure time: 2 minutes 18 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 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
ICC/IF
ICC staining phospho-c-Myc(T58) in SKOV-3 cells (green). The nuclear counter stain is DAPI (blue). Cells were fixed in paraformaldehyde, permeabilised with 0.25% Triton X100/PBS.
FC
Flow cytometric analysis of MCF-7 cells with phospho-c-Myc(T58) antibody at 1/50 dilution (red) compared with an unlabelled control (cells without incubation with primary antibody; black). Alexa Fluor 488-conjugated goat anti rabbit IgG was used as the secondary antibody
IHC
Immunohistochemical analysis of paraffin-embedded human skin tissue with Rabbit anti-Phospho-c-Myc (T58) antibody at 1/500 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 1/500 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.