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How do you quantify your bands? - Chemidoc vs film (Apr/09/2008 )

I am having an interesting discussion wacko.gif with some lab members ...

What is the most accurate way to quantify bands from a western blot for protein expression changes, when you use a chemiluminescent agent?

I have always been told that film is not quantitative because the capture of the image to the film is not linear. Why? The signal actually bounces multiple times onto the film between the intensifying screens. Therefore the signal intensity will change based on multiple factors including distance from the film surface and time of exposure etc.

Thus, I have always performed signal capture from the membrane using either a chemidoc or phospho-image analyser and have been able to get good reproducible results.

I would really like to know what other people think about this issue.




I also often have discussions linked to this issue.

bands detected with film are very nice, but not really quantitative. when you only want to distinguish between protein which is expressed in one sample but not in the other then it is accurate to use films.

I think film is not quantitative, bacause it is not possible for the observer to distinguish between saturated or non-saturated bands. when you expose the x-ray film too long, all bands show the same pattern. e.g. you can expose beta-actin bands for very long time and you will see nice strong bands, but you will not see if you have differences in protein loading.

in chemidoc every photon which is produced during peroxidase reaction is captured by the camera and could be quantified using the program.

hope that helped