SuperSignal West Pico Chemiluminescent Substrate From Pierce

Western blot is a technique that was introduced by Towbin, et al. in 1979; today it is routinely used by many laboratories for protein analysis. The whole procedure involves many steps that must be performed efficiently and with great accuracy. The final step is the detection of the labeled antibody, usually through light emission from a conjugated molecule of Horse Radish Peroxidase (HRP). HRP is activated when it comes in contact with an appropriate substrate and the emitted light is captured by an X-ray film, or by other detecting systems.

SuperSignal West Pico Chemiluminescent Substrate is available from Pierce; it was developed to enhance the detection power of Western blots. During my research project, I had to isolate proteins from the serum of mice after separating them with affinity techniques. I used to test my samples for purity with ELISAs and Western blots. The proteins, after the isolation was complete, were present in the solution at concentrations of about 2 ¥ìg/ml. After the electrophoresis of the samples, I transferred the proteins onto nitrocellulose membranes overnight at 4¨¬C. For the detection of the proteins, I used a primary anti-mouse IgG antibody and a secondary rabbit anti-mouse IgG (dilutions up to 1/50,000), which was conjugated with HRP. The detection of the protein was accomplished by the capture of the light emitted after the addition of luminol-based reagents. I have tried to use at least 2 other kits and the results were quite satisfying. I thought this was the best signal I could get until I tried SuperSignal West Pico Chemiluminescent Substrate. Each blot contained about 0.1 ug of protein, which according to the manual of the kit is too ¡°easy¡± for this substrate to detect.

The SuperSignal West Pico Chemiluminescent Substrate Kit contains a bottle of Luminol/Enhancer Solution (500 ml) and a bottle of Stable Peroxide Buffer (500 ml). In order to prepare the working solution, equal volumes from each must be mixed. I used to prepare 10 ml of working solution for a membrane of about 10 cm x 5 cm. I mixed the solution in a tube which was covered with foil in order to avoid exposure to light. The membrane was incubated in the solution for about 3 min and then a piece of X-ray film was placed on top of it for 30 sec. The first time I used it, I was surprised by the quality of the image on the X-ray film. There was absolutely 0% background and the band, which used to appear faint even after 20 min of incubation with other substrates, was very intense and clear after 30 sec. These results were repeated every time we strictly followed the guidelines of our Western protocols. Today, SuperSignal West Pico Chemiluminescent Substrate from Pierce is my favorite choice for 100% efficient protein detection on Western blots. Furthermore, the solution can be stored at room temperature with no apparent loss in activity even after many months.

Overall, I am very satisfied with the performance of this product and honestly, I haven¡¯t been able to find any major flaws with it. I recommend it to anyone who wants to detect extremely low amounts of proteins without having to worry about the performance of their substrate at the final step of the process, after a long day in the laboratory.