Mouse monoclonal anti actin

Briefly, 50 μg from each sample homogenate was denatured by boiling for 5 min in 2% SDS and 5% 2-mercaptoethanol and loaded into separate lanes of a 12% SDS-PAGE gel. The samples were separated electrophoretically at 100 V for 2 h. The separated proteins were electrically transferred onto PVDF membranes using a T-77 ECL semi-dry transfer unit (Bioscience, Washington, USA) for 2 h. The membrane was blocked in TBS buffer containing 0.05% Tween and 5% non-fat milk for one hour. The membranes were then incubated with either mouse monoclonal anti-NF-κB p65 or mouse monoclonal anti-actin (Santa Cruz Biotechnology, Inc.). Polyclonal goat anti-mouse immunoglobulin conjugated to alkaline phosphatase (Sigma–Aldrich, Chicago, USA) diluted 1:5000 in the 10×-diluted blocking buffer served as secondary antibody. Protein bands were detected by adding alkaline phosphatase buffer (100 mM Tris pH 9.5; 100 mM NaCl; 5 mM MgCl₂) containing the substrate, 6.6 μl NBT/ml and 3.3 μl BCIP/ml (from stock of 50 mg/ml nitro blue tetrazolium (NBT) and 50 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP) in 70% formamide). Colour reactions were stopped by rinsing with stop buffer (10 mM Tris–Cl, pH 6.0, 5 mM EDTA). Relative intensities of protein bands were analysed by scanner and quantified by AIDA Image Analyzer software.