How to quantify & compare protein levels in fluorescently stained cells

Hi there,

I wanted to ask for some input & opinions from you which way might be the most suitable and robust to assess the level of a given protein of interest within cells/nuclei that are detected via immunofluorescence. I want to assess knock down efficiency in cell lines compared with the respective control in the end…

What I did so far, is to measure different intensity values (like mean, IntDen & RawIntDen) of a single focal plane of cell nuclei from the different cell lines but I’m unsure which of those would give the best measure without fearing to underestimate the knock-down efficiency (which was already measured by qRT-PCR, but can of course differ on the protein level), as I need them to make a statement on whether to use them for subsequent experiments …

Thanks for the help! Best,

Patrick