Western blot OD analysis

Posted by Youngmee Sul on Apr 16, 2010; 11:02pm
URL: http://imagej.273.s1.nabble.com/Western-blot-OD-analysis-tp3688571.html

I performed western blot of transfected cos7 cells to determine the levels of our protein of interest. This is our first time using imagej to quantitatively analyze western blot. To test imagej's quantitative capabilities, we loaded 3ul, 6ul, 12ul and 18ul of the same protein in different wells. The gel showed a gradual increase in intensity of the band proportionate to the increase in protein loaded.

I want to determine the densitometry of each of these bands. I used the following procedure:
1. subtract the background with rolling ball radius of 25.
2. outline 1st band (usually start with the brightest band) with box selection tool
3. ctrl + 1
4. outline next band
5. ctrl + 2
6. repeat steps 4 and 5 until all the bands are selected
7. ctrl + 3 (the plots appear)
8. using the straight line tool, I close the bases of all the inverted parabolas.
9. using magic wand, select inside the enclosed parabolas to obtain the areas
10. i used the areas as a measurement of relative intensity.

This method did not yield the appropriate ratios that indicate the correct increase in protein loaded.

What am I doing wrong? Will the areas determined from the gel analysis not reflect the increase in protein loaded? Is there another method to quantify western blots?