A possible cause for concern would be that the anti-Akt antibody, if it is polyclonal, includes some antibodies that recognize only pAkt. This could make the total Akt level appear to increase when actually only the proportion of pAkt increased.

Another possibility is that the expression level of Akt actually did increase.

Hi Shuoshuo, 

Thank you for your reply. 

I am using CHO and HEK cells. 

As for loading control, I am using b-actin, tubulin and ran. They all look pretty even. 

Adam, thanks for the reply!

I am using the most common total Akt antibody from Cell Signal. 

It is polyclonal. 

Perhaps I will try another Akt antibody. 

I am also using gradient gels to run my proteins, could it be a factor?

Thanks again!

Hello Dmitri,

we are using AKT/paKT both from CS on a daily basis in our lab. The total AKT will not increase or decrease with (de-)phosphorylation. Say, if you use an AKT- or upstream inhibitor you will only see reduction of pAKT and not the total which would otherwise be a very bad control. Thus, the increase you see see hints towards a real increase in total AKT protein levels. If it's upregulated you can check my real time PCR. For stabilization/inhibition of degradation other assays will apply.

Hope that helps.

I would echo Adam Shapiro. At it's most basic it could simply be an antibody issue. I know that finding good antibodies can ba a pain, but if another one exists, it might be worth your while to re-run the western with another Akt antibody, should clear things up right away. Another possibility is Shuoshuo Wang's first suggestion. There is a dephosphorylation protocol that can be done after blocking using CIP, I have attached Abcam's version. I have never tried it, but it seems very straightforward. and might save you time and money searching for antibodies.

http://www.abcam.com/protocols/protein-dephosphorylation-protocol

Thank you, Thomas and Dylan, for your helpful suggestions! 

Much appreciated. 

If your antibody stinks like most phospho antibodies and you have a good antibody that recognizes all forms then run on a 2 d gel and transfer it to PVDF.  The phospho group will draw the phosphoprotein away from the non phosphorylated ( separated in 1st dimension pH gradient)).  If you have multiple phophrylated forms you will get several dots at the appropriate molecular weight (2nd dimension)

Hi Dimitri,

I've worked in the past with PI3K/Akt signalling after IGF-1 stimulation. I also saw some times the apparent increase in the band of Akt. At the beginning this was a bit unsettling but then we realized (by blotting less time and with less antibody title) that the big band of Akt was in fact a doublet of bands. Most likely a lower akt and a upper p-Akt both recognized by the total Akt antibody.

Hope it helps