I've noticed this strange phenomenon where my signal intensity will change from picture to picture over the course of my exposure. For example: I will expose membrane to ECL for 2 min, signal will start out medium strength at 5s exposure, then increase in strength at 1 min, continue to increase at 2 min, then drop at 3 min, then increase again at 4 min (now about the same strength as it was at 1 min), then continue to increase until reaching saturation. Maybe my understanding of Chemiluminescence is incomplete but the way I understood it was that the signal should continue to increase (especially when the time scale is minutes) and that should the ECL be used up the signal would begin to decrease. It is the fluctuation that has me confused. To make things stranger, I washed the membrane (3X 5 min TBST) and re-developed with ECL and the results of the second exposure appeared totally different, with reversing trends etc. Any help or explanation of why this is occurring is greatly appreciated.
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