Mouse monoclonal antibody directed against β actin

Hypothalamic tissue samples from rats (95 μg total protein/cell lysate) were loaded and electrophoresis was performed on a 12% gradient Tris-HCl gel (Bio-Rad, Hercules, CA) under reducing conditions. A high protein concentration was used in an attempt to reveal low levels of membrane GR protein in the surface protein aggregate. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes for immunoblotting. PVDF membranes were washed in double-distilled H₂O and blocked with 1% goat serum and 5% nonfat dry milk in TBS-Tween 20 (TBS-T), pH 7.4, for 2 h at room temperature. The membranes were then incubated with a mouse monoclonal antibody directed against a short region of the DNA binding domain of GR (BuGR2, 1:200; Affinity Bioreagents) or a mouse monoclonal antibody directed against β-actin (1:1000, Sigma, Saint Louis, MO) overnight at 4°C. They were then washed 5 times with TBS-T, incubated for 1.5 h with a horseradish peroxidase-conjugated anti-mouse IgG and again washed with TBS-T. The membranes were then rinsed with double-distilled H₂O, immersed in chemiluminescence (ECL) detecting substrate (Amersham Biosciences, Piscataway, NJ) for 3 min, and exposed to HyperFilm ECL film.