Posted by
Joel Sheffield on
Apr 17, 2010; 2:01pm
URL: http://imagej.273.s1.nabble.com/Western-blot-OD-analysis-tp3688571p3688575.html
Gel quantitation is quite complex. First, you have to be sure that your
image is actually one of transmission. I have seen students use images
scanned on a flatbed scanner, in which the light passes through the gel and
is then reflected back through the gel again on its path to the detector
--not good! If you are using a camera to collect the data, you need to know
about the linearity of the camera as well. You also have to be very careful
about linearity of the stain. What about saturation of the stain/film? Is
width/area of the band proportional to the concentration?
Under separate cover, (since this list does not transmit attachments), I am
sending you an exercise that I developed for our cell biology class, using
ImageJ to quantitate a gel. More than anything else, it indicates the
problems that you might face. I agree with Gabriel that applying a
background correction is a very bad idea, since you have very little control
of the level. In this method, rather than isolate specific bands, we scan
horizontally across a concentration series, and then integrate the result,
after adding a "background" line to the graph.
As an aside, one of the interesting approaches to background correction for
an individual band is to calculate the local integrated density under the
edges of the selection box. This method is used in ImageQuant software.
The problem is deciding on the boundaries of the band. I can imagine a
macro to do this, but haven't developed one.
Best of luck,
Joel
On Sat, Apr 17, 2010 at 6:15 AM, Gabriel Landini <
[hidden email]>wrote:
> > 1. subtract the background with rolling ball radius of 25.
>
> Applying "a-posteriori" background correction methods on images that will
> be
> subjected to optical density measurements seems to me a very bad idea.
>
> Cheers
> G.
>
--
Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail:
[hidden email]
URL:
http://astro.temple.edu/~jbs