Okay to use different exposure times to quantitate a western? - (Jul/05/2006 )
Often, when doing a Western blot via the ECL method, when trying to determine the linear range for two or more proteins of interest on the same membrane (i.e. Beta actin plus some other protein of interest...), I often find that, while I am linear on one exposure time for the Beta actin (10 minutes, for example), I am linear on another exposure time (for example, 5 minutes) with the protein of interest.
Is it feasible to use the Beta-actin from one exposure time and "normalize" to a protein band from another exposure time on the same membrane?
I know that it is often a method that, if two proteins are too close together on the same membrane, that you will expose for one protein, strip the membrane, and then expose for the other protein of interest. I think that if it is acceptable to do that (membrane is the same, but obviously exposure times may be different...) then it would be alright to do this, but I am unsure and don't want to proceed if it's a nutty idea.
I appreciate any feedback, and thanks!
you can't do that for 2 reasons.
First, you may overexpose your film... and that's an éliminating condition.
second one : the ECL intensity decreases slowely inducing artefacts in your method.
So 2 alternatives :
an ECL camera which allo a relative good quantification.
Testing different times of exposure, and be sure that over exposure does not occurs. Then scan film an use image J.
The last method gives an idea of ratios, but not an accurate answer.