phospho-protein and total protein significance - (Nov/15/2013 )

Dear scientists,

While doing a western, why does it become irrelevant if the total protein (let's say AKT, eNOS) also change in expression level together with phospho-protein between the two samples? In other words, why should phospho change but total should remain the same while investigating signalling?

thanks,

If your target protein (or enzyme) requires phosphorylation of tyrosine or serine (for example) for activation then an increase in phosphorylation of the protein, without an increase in the expression of the protein itself, is an indication of increased enzyme activity.  If the total protein expression increases along with the phosphorylation state of the protein then there may be many explanations for this, some biological some technical.