I am using the Amersham ECL Western blotting reagent. Following a 5 min exposure, I have no signal. After a 1.5hr exposure, there is a very weak signal. I left film in the cassette to expose overnight. When I came in this morning to develop the film, I was back to absolutely no signal. I was under the impression that this substrate is active for 24hrs. I am using an HRP tagged antibody, but in my experience the HRP has remained active for up to four days.
Depending on which ECL reagent you use, the signal can last for many hours, or just a few minutes.
According to my experience with the Amersham ECL, I think it does not last long. When I do not see bands I used to add ECL again which usually help. Sometimes the reason behind seeing a week band is your primary or secondary antibodies. May be one of them needs to be more concentrated. I would try different percentages of each one on strips and see the results. In addition, if the transferring is not efficient you see no or weak bands. Make sure your protein being transferred efficiently by staining the membrane right after the transferring being done.
Hope this help!
As Adam and Rana indicated, certain ECL solutions do not give a signal that lasts very long. In addition, the signal is not linear. It will peak and then begin to decline. There are some ECL kits that do emit for longer periods of time and are extremely sensitive. One that comes to mind, and it is not the only one on the market, is west femto. These kits can be very expensive relative to the kits you normally use. But, as advertised, they are very sensitive. You may also have to adjust you protein loading and the dilutions of both your primary and secondary antibodies to compensate for the increased sensitivity. Also note that if you are using an antibody with a poor signal-to-noise ratio, a more sensitive visualizing system will not help much.
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