Hi,
I want to determine the pixel density of an immunoblot.
Using ImageJ, I obtained Mean Gray Value and Integrated Density, but neither value really matches with what we observed on the actual image. That is for the 2 areas I selected to measure, they are clearly different by looking at them on the image, but their values are very close to one another.
My questions: Is either mean gray value or integrated density the same as pixel density? If mean gray value or integrated density of area 1 and area 2 differ by 10, for example, how significant is this difference?
Thank you!
I have attached example calculation,
Mean, Int den, RawIntDen - can give relative and same value when you compare lane1 with lane2(Band1 and band2). Area or perimeter should be same for bands we are comparing. You can any prefer one of them for calculations.
Background should be taken with same area for each band (empty space, above or below of band is better=localized background) to avoid improper exposure while imaging.
Finally (1) Test/control=Protein of interest/House keeping gene
(2) Test treatment/Control treatment
(3) Using 3 times repeated blots we can plot graph with std.dev.
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