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I am confused about using the ImageQuantTL application as a tool to analyse the Urea Gel. My problem is that I am not sure if the 'shape' for calculation should be the same for all samples (like on picture A) or as close to the band as possible (like on picture B)? And what about the background subtraction? The `Image Rectangle/Ellipse' option says:

Uses the average pixel intensity in a defined rectangular or elliptical area of the image. To define the area for background calculation, select the background shape in the navigator and then drag with the mouse. Background areas are identified by a broken colored border, and are numbered in the sequence in which they are created. If you have defined several background areas, select the area to use in the Parameters tab.

Does it mean that the shape for the background subtraction not necessarily has to be exactly the same size as the shape for the band? (See Figure C.) I attach some pictures for your convenience. enter image description here

Thank you very much for any help.

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I would definitely go for option A. If you are assuming that the staining in band 6 is all due to whatever you are measuring, there is no reason to think that the signal at the top of lane 1 is not meaningful.

That said, this type of measures is only semi-quantitative, so being too "picky" in how you choose your region probably won't really increase the precision of your result. If you want to get a quantitative measure I am afraid you will have to turn to some other technique.

Finally note that the area obviously means a lot if you are looking at total intensity of the bands, rather than average intensity.

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  • $\begingroup$ I can only agree to tihs answer. Be careful with too "exact" measurements here. $\endgroup$ – Chris Jun 29 '14 at 11:51

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