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I am doing Western blot data analysis and I have probed my membrane with phospho-specific antibodies against a phosphoprotein (it is a protein kinase) of interest. I have also probed my membrane with pan-specific antibodies against the total protein. In my Western blot I am comparing knockout brain lysate samples against wild-type samples. I have also used a loading control.

I want to quantify the levels of my phosphoprotein relative to total protein. The way that I am doing this is that I am calculating the integrated density of my phosphoprotein bands, and dividing that by the integrated density of my total protein bands. However, I was wondering if I need to divide this ratio by the loading control in order for this normalisation to be correct, e.g. (phospho/pan)/loading control?

Is it correct to assume that levels of total protein are consistent between the different experimental groups (wild-type vs. knockout)? Any insights are appreciated.

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