Baseline for Gel Analysis

[ALEX CHAMPAGNE]

I'm currently using ImageJ to analyze thin layer chromatography plates.  It seems that the gel analyzer option is the best way to analyze the density values of my bands.  However, one thing that I've noticed is that the baseline is not often constant or straight when I plot the density profile under the gel analysis menu.  Thus, when I use the wand tool to measure the area under my peak, The tails are often included.  I know that the method used by many people is simply to use the straight line tool to draw their own baseline, but this method makes me nervous, as there are several human errors that could be introduced with this process.  Is there a plugin or existing process in ImageJ that could remove the baseline from the gel profile plots automatically?  My lab group has previously used TN Image to analyze TLC plates, and this program, despite its age, is able to perform such a function.  If anyone has such a plugin, or knows how I might write a macro to generate a baselin
e automatically, I would be greatly appreciative.

Thanks,
Alex Champagne
Ohio State University

[Joshi, Manju]

 I am a new ImageJ user and trying to analyze band intensities in
Coomassie Blue stained gels. I am having same problem (i.e. baseline is
not uniform). It is more of problem if there are 3-4 bands in a lane and
some are dark and others are light. All I am trying to get a relative %
of each band in a lane. Please any tips/advise, I would be greatly
appreciative. Thanks.

Manju

Thanks
-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
ALEX CHAMPAGNE
Sent: Monday, December 10, 2007 9:53 AM
To: [hidden email]
Subject: Baseline for Gel Analysis

I'm currently using ImageJ to analyze thin layer chromatography plates.
It seems that the gel analyzer option is the best way to analyze the
density values of my bands.  However, one thing that I've noticed is
that the baseline is not often constant or straight when I plot the
density profile under the gel analysis menu.  Thus, when I use the wand
tool to measure the area under my peak, The tails are often included.  I
know that the method used by many people is simply to use the straight
line tool to draw their own baseline, but this method makes me nervous,
as there are several human errors that could be introduced with this
process.  Is there a plugin or existing process in ImageJ that could
remove the baseline from the gel profile plots automatically?  My lab
group has previously used TN Image to analyze TLC plates, and this
program, despite its age, is able to perform such a function.  If anyone
has such a plugin, or knows how I might write a macro to generate a
baselin
e automatically, I would be greatly appreciative.

Thanks,
Alex Champagne
Ohio State University

[Jonathan Hilmer]

The new background subtraction process works quite well, but there's
no magic bullet for identifying the separation between signal and
noise.  You can use an automated process such as the parabolic
"rolling circle" now in place, but while manual methods can introduce
human error, such automated ones are susceptible to systematic
artifacts such as the presence of highly overlapping broad signals
(incorrectly identified as background, etc).

Ideally, you would have some sort of perfect correction factor such as
a lightfield image, but that isn't available in many applications.
It's been my experience that minimal use of automated background
subtraction, combined with careful manual methods, gives a very
reproducible result.  Perhaps you could include some example images or
plots that we could give more specific advice.


Jonathan


On Dec 10, 2007 7:52 AM, ALEX CHAMPAGNE <[hidden email]> wrote:
> I'm currently using ImageJ to analyze thin layer chromatography plates.  It seems that the gel analyzer option is the best way to analyze the density values of my bands.  However, one thing that I've noticed is that the baseline is not often constant or straight when I plot the density profile under the gel analysis menu.  Thus, when I use the wand tool to measure the area under my peak, The tails are often included.  I know that the method used by many people is simply to use the straight line tool to draw their own baseline, but this method makes me nervous, as there are several human errors that could be introduced with this process.  Is there a plugin or existing process in ImageJ that could remove the baseline from the gel profile plots automatically?  My lab group has previously used TN Image to analyze TLC plates, and this program, despite its age, is able to perform such a function.  If anyone has such a plugin, or knows how I might write a macro to generate a base!
 lin
> e automatically, I would be greatly appreciative.
>
> Thanks,
> Alex Champagne
> Ohio State University