trouble using ImageJ for densitometry on western blots

[Cozart, Michael A]

I have read the user guide and numerous protocols for using ImageJ to quantify the bands in a western blot.  They all say to use the same size box to measure all the bands.  My problem is that most of our western blots have bands of unequal width.  If I make a box (one size fits all) as per the protocols, either I make the box to fit the narrowest band and the result is that the box does not fit around the wider bands resulting in an inaccurately small read for those bands.  Conversely, if the box is made large enough to fit the widest band, it encloses parts of the adjacent bands when put over a smaller band, thereby giving an inaccurately large read for those bands.  Does anyone have any suggestions?



Thanks,
Michael
Confidentiality Notice: This e-mail message, including any attachments,
is for the sole use of the intended recipient(s) and may contain
confidential and privileged information.  Any unauthorized review,
use, disclosure or distribution is prohibited.  If you are not the
intended recipient, please contact the sender by reply
e-mail and destroy all copies of the original message..

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Blackshear, Joseph L., M.D.]

I have been doing this on VWF multimers.  I use the rectangle function
on the gel, Invert from file menu, then control 1, and control 3 to get
the figure of the bands.  Then selet the verticlal line tool, and draw a
verticle separating each band, being sure that you carry the verticle
all the way to the baseline.  When all peaks are separated, select the
wand tool, and click inside each peak, and results will contain
numerical figures of relative area.
If you find a way of improving on this (getting the line to be verticle
is a little tricky) please let me know.

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
Cozart, Michael A
Sent: Wednesday, June 20, 2012 5:33 PM
To: [hidden email]
Subject: trouble using ImageJ for densitometry on western blots

I have read the user guide and numerous protocols for using ImageJ to
quantify the bands in a western blot.  They all say to use the same size
box to measure all the bands.  My problem is that most of our western
blots have bands of unequal width.  If I make a box (one size fits all)
as per the protocols, either I make the box to fit the narrowest band
and the result is that the box does not fit around the wider bands
resulting in an inaccurately small read for those bands.  Conversely, if
the box is made large enough to fit the widest band, it encloses parts
of the adjacent bands when put over a smaller band, thereby giving an
inaccurately large read for those bands.  Does anyone have any
suggestions?



Thanks,
Michael
Confidentiality Notice: This e-mail message, including any attachments,
is for the sole use of the intended recipient(s) and may contain
confidential and privileged information.  Any unauthorized review,
use, disclosure or distribution is prohibited.  If you are not the
intended recipient, please contact the sender by reply
e-mail and destroy all copies of the original message..

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Gabriel Lapointe-3]

You can also use thresholding and segmentation to select the bands.
   *Gabriel Lapointe, M.Sc.*
Lab Manager
Concordia University, Biology Department
7141 Sherbrooke St. West SP 534
Montréal QC H4B 1R6 Canada
Lab : [phone] x5988
Office : [phone] x3008
Fax : [phone]
Cell : [phone]
[hidden email]
http://gabriellapointe.ca



2012/6/20 Cozart, Michael A <[hidden email]>

> I have read the user guide and numerous protocols for using ImageJ to
> quantify the bands in a western blot.  They all say to use the same size
> box to measure all the bands.  My problem is that most of our western blots
> have bands of unequal width.  If I make a box (one size fits all) as per
> the protocols, either I make the box to fit the narrowest band and the
> result is that the box does not fit around the wider bands resulting in an
> inaccurately small read for those bands.  Conversely, if the box is made
> large enough to fit the widest band, it encloses parts of the adjacent
> bands when put over a smaller band, thereby giving an inaccurately large
> read for those bands.  Does anyone have any suggestions?
>
>
>
> Thanks,
> Michael
> Confidentiality Notice: This e-mail message, including any attachments,
> is for the sole use of the intended recipient(s) and may contain
> confidential and privileged information.  Any unauthorized review,
> use, disclosure or distribution is prohibited.  If you are not the
> intended recipient, please contact the sender by reply
> e-mail and destroy all copies of the original message..
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[oliverd]

ImageJ isn't very flexible with these kinds of things. Check out this paper and see if it answers your question.
Gassmann et al. Electrophoresis 2009, 30, 1845-1855. DOI 10.1002/elps.200800720

[Joel Sheffield]

--------- Forwarded message ----------
From: Joel B. Sheffield <[hidden email]>
Date: Wed, Jun 20, 2012 at 10:35 PM
Subject: Re: trouble using ImageJ for densitometry on western blots
To: [hidden email]


You are absolutely correct in being concerned about the size of the
rectangles that you use.  I also don't see any reference to how you would
correct for background, which can vary considerably from place to place on
a blot.

If you are actually interested in serious quantitation of bands on a
western blot, you might find it instructive to perform a standard curve,
using different dilutions of the same material, and see if any of the
quantitation methods you wish to use are really linear.  I would add that
the method of scanning the blots, whether reflective or transmissive, can
have an enormous effect.

The list won't allow me to send a copy of a pdf file that I use in my Cell
Biology class to introduce some of these principles, but I would be happy
to send it directly to anyone who would be interested..

Joel



On Wed, Jun 20, 2012 at 6:08 PM, oliverd <[hidden email]> wrote:

> ImageJ isn't very flexible with these kinds of things. Check out this paper
> and see if it answers your question.
> Gassmann et al. Electrophoresis 2009, 30, 1845-1855. DOI
> 10.1002/elps.200800720
>
> --
> View this message in context:
> http://imagej.1557.n6.nabble.com/trouble-using-ImageJ-for-densitometry-on-western-blots-tp4999138p4999141.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

--


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




--


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

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html