WB
Western blot analysis of Phospho-STAT3 (Y705) on different lysates with Rabbit anti-Phospho-STAT3 (Y705) antibody at 1/1,000 dilution.
Exposure time: 25 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.
IHC
Immunohistochemical analysis of paraffin-embedded human lung tissue with Rabbit anti-Phospho-STAT3 (Y705) antibody at 1/500 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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.
IP
Cell treatment (CT)
Phospho-STAT3 (Y705) was immunoprecipitated from 0.2 mg HeLa treated with 50ng/mL IFN alpha 1 for 30 minutes cell lysate with Phospho-STAT3 (Y705) Recombinant Rabbit Monoclonal Antibody at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using Phospho-STAT3 (Y705) Recombinant Rabbit Monoclonal Antibody at 1/5,000 dilution. Anti-Rabbit IgG for IP,VHH(HRP) at 1/5,000 dilution was used for 1 hour at room temperature.
Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 24 seconds;