I am looking for phospho-Akt (serine 473) in rat heart tissue samples using western blots. I have done trouble shooting for weeks but still nothing. At first we thought it was our antibodies that were old, but we received new ones and still did not obtain bands. We did however get a band for the positive control. This suggests that there is no p-Akt in my samples, but Akt is so widely distributed that I find this hard to believe. I expect to at least obtain a signal in my control lane.
I have tried blocking in different concentrations of milk and BSA, and I have adjusted the concentrations of my primary and secondary antibodies.
Where could the problem lie?
On the same membrane I have obtained bands for p-Erk and cytochrome c.
The left band in the image attached is the ladder, the right band is the positive control.
Could something in my lysis buffer be interfering?
Please help as this is super frustrating!
As you could detect other proteins in the same sample indicates that your lysate and overall WB tech. is working without any problem. As Adam has already mentioned please check for the total Akt. Again Akt has different phosphorylation site other than ser473. did you check for these sites or you are interested in only ser473 phosphorylation?
Sometimes you have low levels of basal phospho AKT depending on incubation conditions. Maybe you should try a protocol of tissue stimulation (insulin for example).
Hi Toni,
Did you solve your problem with your WB? I'm having the same problem as you but using HepG2 cells stimulated with insulin.
I also thought it was a problem with my old antibody, but similar results were obtained with new antibodies from Cell Signalling (# 9271) and Santa Cruz (# SC81433). I really don't know what to do anymore.
Hi Toni,
I'm having the same problem, while doing some reading found out that if the proteins are not properly denatured, the antibodies won't bind and/or, the proteins will be so "heavy" that wont run through the gel, so you wont be transferring them to the blot, therefore, they are not there.
Have you found a solutions?
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