I'm currently investigating insulin resistance in male and female mice fed a high fat diet.
I am using blunted activation Akt-Ser473 as a marker of insulin resistance.
In this experiment mice are treated with (or without) insulin before being sacrificed, lysates are generated and blotting for total Akt or pAkt.
Using the anti-Akt antibody I see a series of bands at the predicted molecular weight (60kDa). Blotting for pAktSer473 I see two series of bands, one at 60kDa, the other around 75kDa (see image). pAkt is predicted to be 60kDa however the pattern of what I expect to see in insulin treated mice appears at 75kDa.
Once of my colleagues thinks that the bands at 75kDa are non-specific and that pAkt is at 60kDa, while another one of my colleagues thinks that the bands at 60kDa are Akt while the pAkt bands are at 75kDa.
Can someone who has worked with pAkt westerns blotting provide some insight?