Quantification of proteins in Western Bands - (Apr/06/2011 )
I have a protein in my X-ray film that degrades or disappears over time. I am trying to quantify the intesity of the bands over time for example, by what percentage the protein band decreases before finally disappearing. I am using ImageJ (the one found in NIH), but no luck in actually getting a good graph with the numbers I get. Any help will be appreciated.
I also use ImageJ. It is not as easy as I thought it might be. Do you run a standard together with your sample? That is the best way. I have found that you should try an dilute the sample such that it is not to dark. It appears as though the software reaches a saturation limit quite soon. I normally run 4 dilutions of standard followed by 5 dilutions of the protein. I then draw a standard curve and plug in the values for the sample(s).
I assume you're setting your relative comparison for the 'zero' time point? I'm not sure why you wouldn't be able to get a good graph. If the levels of the protein between your samples differ greatly, then you can do what Jacques suggested and dilute the strongest samples. If you normalize those samples to a loading control, you should be fine. Divide the value of your target by the value of the loading control and you should get a good graph.
I've had good results with ImageJ for western blots, HUVEC migration, and cell counting. I follow this tutorial for western blot densitometry: http://howtowesternblot.net/quantification/