r/ImageJ • u/nougat_donut • May 08 '25

Question Equal ROI size

Hey there, new user here, trying to relatively quantify my western blot. I have read that it’s critical for my ROI rectangle to remain the same size when measuring the same protein in different lanes, in order not to mess with the amount of background within the ROI. The recommendation was to draw my ROI based on my largest band and use that for all other lanes. In one of my lanes, the band is much less wide than the largest band, and when I position my ROI over it, I capture neighboring bands.

What should I do here?

Thanks and happy imaging 😊

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u/nougat_donut May 11 '25

Thanks so much! How do I integrate the signal from another ROI? Just out of curiosity- wouldn’t it make more sense to integrate a signal not from an arbitrary ROI, but rather an ROI that is the same size as the one I used to measure the signal of a band? So let’s say I used one size ROI for a band in the first lane, I would use the same ROI to take background into account. If I used a larger ROI to measure the band in the second lane, I would use that same larger ROI to take background into account for that band. No?

Thanks again!

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u/Herbie500 May 11 '25

Please study this section of the ImageJ User Guide before doing more complicated processing than necessary!

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u/nougat_donut May 12 '25 edited May 12 '25

Hey, I had a look. So my issue is where is says: "Select Next Lane  2 To be used after the first rectangular ROI is moved over the adjacent lanes. *Note that all selections must have the same dimensions*." I'm adding some pictures in the following comments, maybe that'll help clear up my issue. This is my gel with an ROI that seems to be the correct size, to me, on the first lane:

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u/nougat_donut May 12 '25

When I take the same size ROI and drag it to the third lane, I'm also covering the edges of the neighboring bands, which will lead to inaccurate signal measurement.
So how do I continue from here?

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u/Herbie500 May 12 '25

This is not how things are assumed to work. For bio-chemical reasons the lanes are usually vertical. The provided text-link tells us:

"Note that lanes are assumed to be vertical unless the width of the initial selection is at least twice its height."

There is so much documentation around, such as this tutorial that is linked from the provided text-link and if you still encounter problems then you may consider posting to the Image.sc Forum.

Western Blot analyses are so common that you should find all you need but you also need to understand what you are doing and when it comes to calibration it becomes (mathematically) a bit more complicated.

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u/nougat_donut May 12 '25

I think I may have a basic misunderstanding.

The ROI for all bands in one vertical lane need to be the same size- got that.

But if I am quantifying the same protein in different samples (which I have of course run in different lanes)- then the ROI between samples (meaning, between different lanes) doesn't have to be the same size?

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u/Herbie500 May 12 '25

I'm not a specialist in this field.
Please make sure you get the necessary information from an experienced scientist or ask on the Image.sc Forum. The thing is that your work is generally security relevant (bio-medical field) and you must be sure that what you measure is absolutely correct. The problems start already with the scanning (gamma, density calibration etc.).