There are several P-Akt antibodys you can use, but I see the best results with this antibody from cellsignaling: Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060

CS XP antibodys are their best abs.

If you get a high background or many unspecific bands, you can try to add more Tween20 (up to 0,2%) and a little SDS (up to 0,02%) to your secondary antibody solution. Additionaly, you can reduce your secondary ab incubation time (max. 30min) or use a higher ab dilution (usually you can dilute your abs 2 to 4 times higher then the distributer advises).

Good luck!

Well, i have the same problems with this antibody.

In our lab we work with the pAkt antibody from cell signaling and sometimes it works and sometimes it does not. We also tried to change the conditions (blocking with BSA 5%, blocking with milk) but both are realy not the fact, that this AB works like it wants. After adjusting the methods, I decided for me to incubate the nitrocellulose membranes for 48h with the pAkt - AB. The effect was much better compared to shorter incubation. So probably you just adjust your method by incubating for a longer time.

In my case, p-AKT (S473) has been incubated with membranes at 4oC overnight. Usually it works well. To detect low level of p-AKT, 1:1,000 dilution or less may be helpful.

There are several P-Akt antibodys you can use, but I see the best results with this antibody from cellsignaling: Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060

CS XP antibodys are their best abs.

If you get a high background or many unspecific bands, you can try to add more Tween20 (up to 0,2%) and a little SDS (up to 0,02%) to your secondary antibody solution. Additionaly, you can reduce your secondary ab incubation time (max. 30min) or use a higher ab dilution (usually you can dilute your abs 2 to 4 times higher then the distributer advises).

Good luck!

I have used #9271 without problem for human breast cancer cell lines.

Use the secondary Ab-HRP as little as possible (e.g., 1: 10,000) so that non-specific bands most likely coming from contaminating blood plasma proteins in your brain lysates that cross-react with anti-rabbit IgG-HRP. An alternative is to pre-incubate with a similar anti-rabbit IgG prior to P-Akt incubation.

I have used this antibody from cell signalling though only on cell lysates so far. I use 5% BSA to block and sometimes when you get dirty blots, it is worth while to try just 1-2 hr primary antibody at room temp instead of overnight in the cold. I have done this for other proteins on tissue lysates and it has given me cleaner westerns. Hope it helps.