I am attempting to quantify the relative signal intensities of various bands from western blot images (essentially as described here: http://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/).

However, when adjusting the levels of the minimum signal values under the B&C window to visually identify various bands of differing intensities (e.g. different protein antibody signals) the resulting signal intensity profile plots vary.

When opening a greyscale .tif image of a western blot I first adjust the minimum level with the brightness & contrast menu to a point at which visual identification of the desired band is optimal. Then create a rectangular ROI over the band of interest and continue on in the same manner for each of the subsequent sample lanes (via using the 1, 2, 2, 2, ... commands). After this point I use the 3 command to create the profile plots and use analyze>gels>label peaks to have Fiji integrate the peaks and produce percentages. The issue comes up with the necessity to readjust the B&C of the image to analyze additional bands. When creating a higher threshold minimum in the B&C menu to visualize lower intensity bands FIJI seems to measure values from that new image. (e.g. recording higher amplitude and/or area signal intensity traces).

I was under the impression that adjusting the minimum values for the B&C only altered the display qualities (visualization) of images and not the data upon which FIJI draws to manipulate for output. When completing this same process on a labmate's computer (a macbook pro) the same problem does not occur (e.g. after adjusting the levels the resultant signal intensities from the ROI are identical to before adjustment), so it may be within the version I am using. I have uninstalled and re-installed FIJI as well as ensured that I have updated to the latest build version with no success.

Please let me know if there is anything you reccomend or if you have any more information on this potential bug. I apologize if this is an erroneous submission but after not having the same issue on another machine/FIJI program I have run out of ideas for fixing the issue. Thank you.