The GE Healthcare ECL Western blotting system is a chemiluminescent, non-radioactive method to detect antigens that have been immobilized onto membranes. This system makes use of a horseradish peroxidase (HRP) conjugated secondary antibody that, in conjunction with a chemiluminescent substrate, luminol, generates a signal that can be captured on film.
I have been using this Western kit for two years now, and have favored this system over other detection methods. The great advantage of this kit is that, with our experiments, we can re-probe the Western blots many times to look for other proteins with very little loss of signal. With other detection methods, such as NBT/BCIP or DAB based methods, a colored precipitant forms on the blots, which makes re-probing difficult to impossible. Other chemiluminescent kits claim to allow re-probing, but special stripping buffers are usually required which can then lead to a loss in signal intensity. Our experiments often involve immunoprecipitations from precious samples, and re-probing western blots allows us to make these samples go farther.
Another advantage of the ECL Western kit is how long the signal lasts. Most of the time I get the exposure I need in less than five minutes, but in cases where a protein may not be very abundant in the sample we are probing, a signal that lasts many hours is beneficial. Because the signal from the ECL kit can last up to 24 hours, I’ve often left the film on the blot for several hours (or even overnight) for a weak signal and been able to detect it. With many other kits the chemiluminescent signal may only last for a few minutes. I’ve also found this helpful when my boss decides (three hours after I’ve developed the blot) that a shorter exposure might give us the perfect figure.
GE Healthcare has also developed many accessories to the ECL kit. This includes a variety of HRP conjugated secondary antibodies, special membranes that increase the sensitivity of the signal (and increase the ‘re-probability’ of the blot), ECL Western Molecular Weight Markers, Hyperfilm ECL (which is supposedly 30% more sensitive to the signal that is generated by ECL) and even an ECL camera, the ECL Mini-Camera, that allows westerns from mini-gels to be captured directly on Polaroid film. Because this product is quite stable and I’m happy with the sensitivity that I achieve using this system, I feel that this system is definitely worth the money (I’ve used other kits that cost more that had less sensitivity and shorter signal duration). In addition, you don’t have to worry about special membranes (although they can increase sensitivity). Our lab routinely uses nitrocellulose, one of the cheapest membranes that can be used for Western blotting, and I have re-probed the same blot up to five times with very little loss of signal.
The GE Healthcare ECL Western Blotting Detection kit has worked out well for our lab. For blot re-probing, expense and sustained time of signal, I have not found a better kit.
Cynthia Vied
Graduate Student
University of Alabama @ Birmingham