When analysing phosphoproteins via Western blot, why is total protein level of the target protein recommended as an internal loading control?

Question

I am analysing the expression of a protein kinase via Western blots, and it is a phosphoprotein. I have labelled my membrane with antibodies against the phosphorylated form of the protein (using phospho-specific antibodies) and also for total protein (using pan-specific antibodies).

I have read that when studying phosphoproteins, you should normalise the phosphorylated protein signal to the signal for total protein. In this journal paper, it states that the Journal of Biological Chemistry recommends that “signals obtained using antibodies specific for phosphorylated epitopes should be normalized to the total protein level of the target protein”.

I have seen in other journal papers analysing the expression of a phosphoprotein via Western blots that they also normalise the phosphoprotein signal to total protein signal (of the target protein).

However, I am not sure why the total protein level of the target protein can be used as an internal loading control?

I have read in this handbook that internal loading controls are endogenous reference protein(s) that are present in all samples at a stable level and unaffected by the experimental conditions.

I want to compare the expression of my phosphoprotein between samples of different experimental conditions. I know that normally total protein stains such as Ponceau S or housekeeping proteins are used as an internal loading control and for normalisation.

But why is it recommended that you should normalise to total target protein (when looking at phosphoproteins), because one does not know whether the expression of total target protein will be stable between different experimental conditions? Any insights are appreciated.

Answer 1

It is because you don't know how your treatments affect the expression of the total specific protein; perhaps it down- or up-regulates the amount of protein being made, but the relative proportion of phosphorylation doesn't change. For example:

Imagine you have 4 samples from different treatments of which you have loaded an equal amount of total protein (lets say 50 ug total, note that this is too much to load on a WB for accurate quantitation; usually you want less than 10 ug). You run a western blot and find the following numbers (completely made up BTW) for the specific protein. In this scenario, 1 is untreated control.

1. 10 ug
2. 20 ug
3. 10 ug
4. 20 ug

You then try the phospho-specific antibody and get the following

1. 1 ug
2. 2 ug
3. 3 ug
4. 1 ug

What does that tell us about the 4 treatments?

Well, it tells us that 2 is up-regulating the total protein, but the relative proportion of phosphorylation is the same. In 3 phosphorylated protein is up-regulated (3x) but not total protein and in 4 total protein is up-regulated but phosphorylated is down-regulated (0.5x).