film saturation in immunoblots - (Mar/13/2014 )

At a recent conference in which I presented some data from immunoblot experiments I was asked a question for which I didn't have a good answer.  I was asked how did I ensure that the film that I was using to develop the westerns was showing bands in the linear range of the film?  The person asking the question thought that some of the bands shown on the westerns appeared to be saturated. All I could say was that we exposed the film long enough to see a band in our control sample.  I couldn't really answer the question.  Can anyone help me with this? How can you ensure that you are in the linear range of the film when you are exposing a western?

thanks.!
 

The answer to this is that you need to ensure that the film is not saturated, which does indeed invalidate any quantitation you might have attempted.  Saturation is where the film is maximally exposed and can get no darker, after this point the film is no longer in its linear range.  It is difficult to assess at which point an individual blot will show saturation, as it depends on a number of different factors, but the best bet is to do multiple exposures and see.