I would check the type of antibody you are going to use against total AKT.  

The antibody against phospho-AKT should be a monoclonal antibody that binds to the ~5-10 aminoacid sequence that contains the phospho-residue.  

If the antibody agains total AKT binds somewhere else there shouldn't be any competition issues.  If the anti-AKT is  a monoclonal you find the sequence it binds to and corroborate it is different from the sequence the anti photo-AKT binds to.

If the anti-AKT antibody is a polyclonal I wouldn't worry about it.

Ideally I would use both monoclonal antibodies, so the binding stoichiometry  Ab:protein is 1:1.   If one of the antibodies is polyclonal then you could have multiple antibody molecules bind the same antigen molecule and this would give you a stronger signal compared to the signal from the monoclonal antibody, and you couldn't compare both signals quantitatively.

I would also make sure the ammount of antigen is the limiting factor and the Ab is in excess (which usually the case). To make sure you saturated the binding of the Ab.

Good luck !

Fabian

I would check the type of antibody you are going to use against total AKT.  

The antibody against phospho-AKT should be a monoclonal antibody that binds to the ~5-10 aminoacid sequence that contains the phospho-residue.  

If the antibody agains total AKT binds somewhere else there shouldn't be any competition issues.  If the anti-AKT is  a monoclonal you find the sequence it binds to and corroborate it is different from the sequence the anti photo-AKT binds to.

If the anti-AKT antibody is a polyclonal I wouldn't worry about it.

Ideally I would use both monoclonal antibodies, so the binding stoichiometry  Ab:protein is 1:1.   If one of the antibodies is polyclonal then you could have multiple antibody molecules bind the same antigen molecule and this would give you a stronger signal compared to the signal from the monoclonal antibody, and you couldn't compare both signals quantitatively.

I would also make sure the ammount of antigen is the limiting factor and the Ab is in excess (which usually the case). To make sure you saturated the binding of the Ab.

Good luck !

Fabian

I agree with Fabian on most point, although phosphor-antibody are most of the time also polyclonal antibody. The bottom line is both antibodies should not share the same epitope. Like Fabian mentioned, your antibody should always be in excess or else your WB won't be semi-quantitative. You should performed an antibody dilution curve (by increasing the antibody concentration, your signal should eventually saturate). 

Sorry, but I am not competent to answer your question. Fritz Schröder

Constantinos brings what I was going to say. If you have two species of origin, as well as different epitopes, you should be able to probe both with flourescent western.  However, if you are using the IREdye, you can get one conjugated with the 680 wave and the other at the 800 (for different colors) this will also enhance the different protein states.

You can do sequential staining if you have concerns; first the phospho up to the secondary, then the total. Another option is to run the phospho entirely and then reprobe, if you don't care to get both in the same picture. In  this case, you can run the phospho entirely, strip the membrane and reprobe with the total.

You've gotten many great suggestions. At LI-COR we routinely do this type of total-phospho WB analysis. We've run a variety of controls to look for an "antibody interference" phenomenon in this situation, and have rarely seen any effect. Multi-color total/phospho experiments are greatly preferable to blot-strip-reprobe approaches.

It's important to remember that this experiment will NOT give you phosphorylation stoichiometry information. Even if both Ab's are polyclonal, the antibody affinities will be different.