As my protein levels appears to be varying in different cell types and different layers and localization (cytoplasm/nucelus) of the root tip of Arabidopsis (in the background of Wild type and mutant plants).
I wonder what should be my approach to compare differences in protein expression levels and localization between two genotypes.
I take Z-stack in a confocal microscope, usually I make a maximum intensity profile of Z- stack and try to understand the differences but as the differences are not only in intesities but also cell types and layers in that case how should I choose the layers between two samples?
My concern is how to find out exact layers between two genotypes as the root thickness is not always same and some z-stacks for example have 55 slices and some have 60.
thanks!
Avinash Sharma
To compare differences in protein expression levels and localization between two genotypes in Arabidopsis root tip, you can consider the following approaches:
It is important to use a rigorous and systematic approach to ensure that the results are accurate and statistically significant.
Hi, the answer provided by Prof. Janak Trivedi is pretty comprehensive, agree with that. The ideal approach would be to capture equal number of slices for each stack, but I guess some samples have the signal spread over a greater depth (axially) so you don't want to miss out that signal. Also, you mentioned you make "a maximum intensity profile of Z- stack". So I suggest you average out and also make a montage of your stacks (ImageJ options) and then compare the intensity profiles. Additionally. check out this article:
Article A Robust Co-Localisation Measurement Utilising Z-Stack Image...
Hope it helps.
Thank you very much Professor Janak Trivedi for the wonderful suggestions. I will read further to see which method I could utilize in my study.
Thanks a lot to you Arti Tyagi for the idea about making montage and comparing intensity, I will go through the research article you attached.
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