Amersham ECL Prime Western Blotting Detecting Reagent from GE Healthcare Life Sciences

Protocol Overview

After resolving the protein (50ug/lane) on a SDS-PAGE (mini-gel), the proteins were transferred onto nitrocellulose membrane. The membrane was blocked with 5% fat-free milk for 1 hour at room temperature and incubated with 1:1000 dilution of the primary antibody in 5% BSA overnight at 4°C. The blot was washed with Tween-Tris-buffered saline (TTBS), 4x, 15 minutes each and then incubated with 1:1000 dilution of HRP-conjugated secondary antibody (Goat anti-rabbit IgG (H + L)-HRP conjugate, from Bio-Rad cat #170-6515) for 1 hour at room temperature. The blot was washed 4x with TTBS as before. For chemiluminescent detection, 1 mL of ECL Prime Western Blotting Detection reagent 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 5 minutes. 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 x-ray film cassette. X-ray film was placed over the membrane and exposed for 1 , 3 and 5 minutes, and then the film was developed to visualize the signal.