Band area calculation for western blot quantification

Posted by Mamenko, Mykola on
URL: http://imagej.273.s1.nabble.com/Band-area-calculation-for-western-blot-quantification-tp5000025.html

Hello everyone,

I recently started to use ImageJ to quantify my blots and noticed a weird thing about the band area calculation.

To my understanding ImageJ should return similar values of the band areas, regardless of the number of lanes being plotted/selected simultaneously (given the area of selection rectangular is the same).

Perhaps, my particular case would be more illustrative. The blot for quantification had 12 lanes in it. I used a rectangular selection tool (the dimensions of the rectangular were 145x65 pixels). When only the first lane was selected and plotted, ImageJ would return the area of the band as ~54k. But if all 12 lanes were plotted (with the same rectangular tool dimensions), the calculated area of the band in the first lane dropped to ~35k. The less lanes are plotted/selected - the closer calculated first band area is to the 1 lane selection measurement.

The background was homogeneous for all lanes.

Thank you for you consideration. Let me apologize for potentially unwise questions raised here. Any comments would be highly appreciated.

Best regards,
Mykola Mamenko.

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