Primary mouse anti oct 3 4 sc 5279 antibody

Untransfected NCCIT cells were lysed with 1 mL of lysis buffer (1% Triton X-100, 5 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HC1, pH = 7.4, 150 mM NaCl, and 0.1% sodium dodecyl sulfate (SDS)), 1% protease inhibitor cocktail, and phosphatase inhibitor (Sigma). Protein concentrations were measured by the Bradford Protein Assay reagent. Briefly, 20 µg of cell lysates were isolated on 12.5% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). Blocking was carried out with 5% skim milk in Tris-buffered saline (TBS), containing 0.05% Tween 20 (TBS-T). The membrane was then incubated for 1 h with primary mouse anti-Oct-3/4 sc-5279 antibody (Santa Cruz Biotechnology) diluted in 3% blocking buffer and washed with phosphate-buffered saline (PBS) for 30 min at room temperature. A secondary horseradish peroxidase (HRP)-conjugated sheep-anti-mouse antibody (Avicenna Research Institute) was added next and incubated at RT for 1 h. After washing, signals were detected using the Amersham ECL Prime Western Blotting Detection kit (GE Healthcare Life Sciences) and quantified by Gel Logic 2200 (Kodak).