I am new to Western Blot analysis and I have recently done my first two. I am studying a phosphoprotein (a protein kinase) that can be both activated and inactivated via phosphorylation at a specific amino acid residue. I have labelled my membrane against the active and inactive forms of my protein of interest (using phosphospecific antibodies), as well as the total protein (using pan-specific antibodies).
I know that Western Blots are used to quantify the expression of a protein (i.e. how much of a specific protein there is in a sample). But I was wondering how they can be used to quantify the activity (e.g. how functional the protein is) of a protein, e.g. how can I measure/determine whether my protein is more active/inactive in my treatment samples vs. control?
For example, I have normalised the integrated density values for my active protein bands against the integrated density values for my total protein bands. Then I have calculated the fold change in protein level for my active protein in treatment samples relative to control.
If I have observed that the fold change for my active protein is reduced by 40% (as an example) in treatment samples relative to controls, would this be sufficient data to say that the activity of my protein of interest is reduced in treatment samples compared to control?