Western blot analysis of Nanog on different lysates with Rabbit anti-Nanog antibody (ET1610-2) at 1/2;000 dilution. Lane 1: NCCIT cell lysate Lane 2: HeLa cell lysate (negative) Lysates/proteins at 20 µg/Lane. Predicted band size: 35 kDa Observed band size: 40 kDa Exposure time: 3 minutes; 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 (ET1610-2) at 1/2;000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50;000 dilution was used for 1 hour at room temperature.
ICC staining of Nanog in F9 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 (ET1610-2; 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).
Immunohistochemical analysis of paraffin-embedded human seminoma tissue with Rabbit anti-Nanog antibody (ET1610-2) at 1/800 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 (ET1610-2) at 1/800 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.
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either Nanog (ET1610-2) or Normal Rabbit IgG according to the ChIP protocol. The enriched DNA was quantified by real-time PCR using indicated primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin; which is equivalent to one.
