Problems with densitometry!....for WESTERN BLOTS

[Paul D. Boyer]

gjmadlam at umich.edu (Gerard ) writes:

>Hi,

>I've just made a strange discovery when developing by blots.  I found that
>when I expose my blots to film, after using ECL, the bands that I get for
>shorter exposures aren't always "lighter" than the bands I get for longer
>exposures.  They are sometimes darker!!!!  This also happens in reverse
>(of course).  I was just wondering if anyone else has had this problem
>and, if so, what solutions are there.  Thanks.

>Gerard

Gerard- I got past the point of pulling my hair out trying to quantitate
westerns by ECL.  I asked for help some time ago, as well, and the 
overwhelming response was that this is a futile pursuit.  Obviously,
someone has made it work, since Amersham published a dose response
curve in the lit.  I think only a couple people felt confident with it,
and I wish you the best of luck.  There's always 125I and fluorography!

                               Paul D. Boyer, Ph.D.
                               pboyer at mizar.usc.edu
                    Assistant Professor of Biological Sciences
                         University of Wisconsin-Parkside
                              900 Wood Road, Box 2000
                              Kenosha, WI  53141-2000