Hey Caroline!

I faced similar problem during densitometry using ImageJ software. In that situation after the acquisition of data you will have peaks for all the bands, in the case of overlapping bands you will get two peaks, You can draw a line joining two ends of biggest peak to quantify it, smaller peak is for overlapping band leave it as it is. In this way you will get results for your band of interest only.

You could use the smallest band as your box size, effectively normalizing everything to that band. Your positive control band will then max out, but everything will make sense as relative to the smallest band. Alternatively, you can manually quantify each individual band and use that same box size (per band) for your loading control image (Ctrl+Shift+E) instead of doing everything at once.

I am not entirely sure what you mean by manually quantifying each individual band, sorry. 

Normally I draw a box around the biggest band, then hit "1", then move it to the next band, hit "2" and so forth until I've boxed all the bands, then hit 3 to get the individual peeks.  

As long as I have to use the same box size I don't think it will work to quantify them individually.

I also don't see how using the smallest band will normalize everything if all the other band only have partial areas because they fall out of the box?

I've seen people use 1 horizontal band across all the bands and then do "plot lanes" but that gives peaks that are not clearly defined per band so I don't know where to cut it off to get to correct areas for the right peaks.

Thank you for your help though!

Hey Caroline!

I faced similar problem during densitometry using ImageJ software. In that situation after the acquisition of data you will have peaks for all the bands, in the case of overlapping bands you will get two peaks, You can draw a line joining two ends of biggest peak to quantify it, smaller peak is for overlapping band leave it as it is. In this way you will get results for your band of interest only.

@Jordan R. Yaron the procedure you mentioned seems useful but its not clear, please make it more clear by your explaination.. Thanks

@Shivani Dixit, this might be a suitable point in dealing such issues. Thanks

As you said, you can simply use a horizontal rectangle to include all the bands.

Looking at your western image, if the bands are not linking with each other, there should be obvious trough at the empty area between the two bands, and you can draw boundaries to isolate the only the significantly higher level area.

Alternatively, you could also give a try on another software--Image studio lite. You can manually draw rectangles with different sizes to quantify individual bands and also some more functions with this software. You can get the software free in this link:

https://www.licor.com/bio/products/software/image_studio_lite/

Thanks Man Hei Cheung for you brilliant answer its really useful.

Thanks Man Hei Cheung !

I will surely try this method and I hope it will be useful for me. 

Best

I usually draw a rectangular box covering all my bands and hit CTRL1 then CTRL3 to plot lanes. this will give you peaks where you have bands and the space between the bands in negligible background (not a peak). Draw straight lines use the tool bordering your gel bands. Use the wand tool an click on your peaks, next label peaks, this will give you your numbers. You can use theses numbers for your quantification analysis further.