Anti gapdh polyclonal antibody

Human HEK293, H1299, and K562 cells, and mouse RAW264.7 cells were collected and lysed 48 h after transfection to extract total proteins. The cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) using a Bioruptor sonicator. Proteins were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, blocked for 1 h in TBS containing 0.1% Tween 20 surfactant and 5% (wt/vol) nonfat milk with rocking at room temperature, and then incubated overnight at 4 °C with the following primary antibodies: anti-Phospho-NF-κB p65 rabbit monoclonal antibody (1: 1000), anti-NF-κB p65 rabbit monoclonal antibody (1: 1000), anti-Phospho-IκBα mouse monoclonal antibody (1: 1000), anti-IκBα rabbit polyclonal antibody (1: 1000), anti-CTCF rabbit monoclonal antibody (1: 1000), anti-Myc tag mouse monoclonal antibody (1: 1000), anti-HA tag rabbit monoclonal antibody (1: 1000) (all from Cell Signaling Technology), Anti-BORIS/CTCFL antibody, clone 4A7 (1: 500) (Millipore, USA), and anti-GAPDH polyclonal antibody produced in rabbit (1: 5000) (Sigma-Aldrich). The secondary antibodies used were goat anti-mouse (H+L) HRP and goat anti-rabbit (H+L) HRP (both from Dawen Biotec).