I'm quantifying proteins on western blots, using chemiluminescent ECL primer and a CCD-based imager (Carestream) to visualize the bands. I can use exposure times of seconds to minutes. The shorter exposure times give greater dynamic range between "low" and "high" signal bands, but much more noise on the densitometric plot (on both peaks and background, via ImageJ). Longer exposure times give very nice looking bands, no noise on densitometry, but a lower dynamic range. Looking at the histograms, the longer exposure times don't seem to be over-saturated, with no pixels showing at the greatest intensity.
Is there a way to determine the best exposure time? Am I on the right track, looking at the histograms, etc?
Any suggestions would be greatly appreciated.
Hi, Brad, you question is very interesting, indeed. As I understood, you want to find relkative differences in the protein contents in 2 samples, right? First, you to avoid saturation in the long exposure time. Second, you can try to find relative ratio between 2 samples at different exposure time and use the range when you have similar ratio between band intensity. In addition, you can try load different protein amount on the track in order to see how you measure is reliable. For example, 2, 5 and 10 µg of the protein to each track. This will give an additonal basical point for comparison. Good luck!
I would suggest diluting your ECL in PBS if it is very strong. This will let you use longer times without getting over saturated bands. I also try to keep the blots covered when you treat them with ECL. This will give you more consistent results.
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