Horseradish peroxidase hrp conjugated goat anti rabbit

Primary antibodies used for Western blot analysis are the following: rabbit anti-p-STAT-3 (1:1,000, Cell Signalling, 9145S, Cell Signalling Technology, Danver, MA, USA), rabbit anti-STAT-3 (1:1,000, Cell Signalling, 4904S), mouse anti-β-actin (1:5,000, Sigma-Aldrich, A5441, Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies used for Western blot analysis are the following: horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:1,000, Dako, P0448, Agilent Technologies, Santa Clara, CA, USA) and goat anti-mouse (1:1,000, Dako, P0447).

Cells were lysed in RIPA buffer (50 mM TRIS, 150 mM NaCl, 0.5% (v/v) NP-40, 1% (v/v) Triton X-100, 0.25% (w/v) Sodium deoxycholate, 1 mM EDTA pH 7.4, 0.5 mM EGTA pH 7.4) on ice for 30 min. Lysed cells were centrifuged at 10,000 x g for 10 min to remove debris. Protease inhibitor cocktail (1:100 dilution; Sigma Aldrich) was added to cleared lysate. Total protein was quantified using BCA assay (Merck, Germany) or DC protein assay (Biorad).
Protein (25 μg) was fractionated by electrophoresis through 4–20% (w/v) SDS-PAGE precast gels (Thermo Scientific, UK) and transferred to nitrocellulose membranes. Membranes were incubated in 5% (w/v) non-fat powdered milk (Marvel, Premier Foods, UK) in TBST (10 mM Tris-HCL, pH 7.4, 100 mM NaCl, 0.1% (v/v) Tween-20)) for 1 h. Membranes were washed in TBST then incubated in primary antibody diluted in blocking buffer for 16 h at 4 °C (1:4000 rabbit anti-IGFBP4 (Millipore, Ireland), 1:2000 rabbit anti-Akt (Cell Signalling Technologies, USA), 1:2000 mouse anti-pAkt (ser 473; CST, USA). Membranes were washed 3 times in TBST then incubated in 1:2000 Horse-radish peroxidase (HRP)-conjugated goat anti-rabbit (Dako, Denmark) or goat anti-mouse antibody (Dako, Denmark) 5% (w/v) non-fat powdered milk TBS-T for 1 h. Membranes were washed 3 times in TBS-T and antibody binding was visualised using ECL reagent (Thermo Scientific, UK).