Re: Quantification problem: more values despite weaker band?

Posted by Rena2014 on Apr 28, 2014; 7:02pm
URL: http://imagej.273.s1.nabble.com/Quantification-problem-more-values-despite-weaker-band-tp5007430p5007476.html

Dear Joel,

Fortunately, we use an LAS imaging system from Fuji to develop our gels, though there is no need for an extra scanning. But nevertheless, one should definitely be more careful andbetter  think about protocols before just using them...  Thanks anyway!

Verena

2014-04-28 19:45 GMT+02:00 JOEL B. SHEFFIELD [via ImageJ] <[hidden email]>:

Hi Rena,

This protocol has some serious errors in it.  However, the most glaring is
that the scanning is done on a "conventional flatbed scanner".  You might
be aware that the light in a scanner will actually pass through your
intermediate bands twice--once as it leaves the light source, and again
when it is reflected from the back side of the scanner.  As a result, your
readings might not be particularly quantitative.  Some scanners have
specific modes for transmission.  It's much better to use one of those, or
even a carefully done photograph.  In all cases, I would suggest that you
carry out a true calibration of bands to ensure that you are getting linear
results.

Joel






Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: <a href="tel:215%20204%208839" value="[phone]" target="_blank">215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs

On Mon, Apr 28, 2014 at 9:40 AM, Rena2014 <[hidden email]>wrote:

> Hi,
>
> Thanks already for your comments on my quantification problem!
> So far I followed a protocol from the University of Missouri
> (
> http://support.dalton.missouri.edu/index.php/wiki/Public:Quantifying_Color_Intensity/
> ).
> As recommended, I checked the "true intensities" and in some of my images I
> see high values for the black bands and low values for the white background
> (if the image is not inverted). So, clearly here is something inverted,
> right?
>
>
>

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