Pierce ECL Western Blotting Substrate from Thermo Scientific

Protocol Overview

After resolving the protein (50 ug/lane) on a SDS-PAGE (mini-gel), the proteins were transferred onto nitrocellulose membrane. The membrane was blocked with 5% dry milk for 1 hour at room temperature and incubated with 1:10,000 dilution of the ?-actin antibody in 1% dry milk overnight at 4°C. The blot was washed with Tween-Tris-buffered saline (TTBS), 4x, 15 minutes each and then incubated with 1:20,000 dilution of HRP-conjugated secondary antibody (Goat anti-mouse IgG (H + L)-HRP conjugate; Bio-Rad cat #170-6520) for 1 hour at room temperature. The blot was washed 4x with TTBS as before. For chemiluminescent detection, 1 mL of Pierce ECL Western Blotting Substrate Solution A (luminol reagent) was mixed with 1 mL of Solution B (peroxide solution), mixed and overlaid onto the nitrocellulose membrane, which was sitting on a piece of Saran Wrap; the blot was incubated with the detection solutions for 1 minute. The excess reagent was drained by placing the edge of the membrane against a tissue. The membrane was then placed in a clean sheet protector and placed in an film cassette. X-ray film was placed over the membrane and exposed for 1 , 3 and 5 minutes and then developed to visualize the signal.