Akt-P-Ser473 antibody, clone 6F5, Millipore. At least works with PC12 cells...

Hi,

I used the Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb from cell signaling in samples from rat brain (hippocampus, hypothalamus and cortex) and I have detected one clear band in Western Blot. It is maybe the best antibody that exists in the market and it works quite good for IHC too. Maybe there the rat skin tissue is expressing another detectable isoform. Did you try the same antibodies in other type of tissue-cells?

I successfully used the Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb from Cell Signaling in samples from rat livers. It worked fine both for western blotting (1:300 dilution) and immunohistochemistry (1:100) . We detected only one band by western blotting at the expected size. You should try to silence AKT via siRNA and see which of the two bands disappears.

We have used the Cell Signaling antibodies for total and phospho-Akt Ser473 and Thr308 using the dilution recommended by the company to detect phosphorylated Akt in tissues from rat bladders (Ramachandran et al., 2010, Am. J. Pathol. 177(1): 119-131).

I am using pAkt Ser473 #4058 from Cell Signalling and it works very well in primary rat adipocytes and skeletal muscles.

I echo what most have said, the Cell Signaling AKT, both total and phosphorylated, works great.

well, let's not forget, that she actually had problems with Cell Signalling antibody...

Phospho-Akt (Ser473) (736E11) Rabbit mAb #3787

I have used both of them in rat cells and they gave results as expected, one single band (upper one). It seems to me that lanes 1, 2, 3 are reactive and 4 (from lt. to rt.) non-reactive, the lower band seems non-specific. You can do an IP to confirm how many proteins do they bind with. You can also improve the upper band by an extensive washing.

Thanks everyone for your input to the salvation of my little problem here :)

Kchitij, the Abs you've suggested are said to work only for IHC, so did you try them for IB in rat?

I will give a go to Abs from Millipore, CS #4058 and maybe Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific) #9018

Try the Rab mAb, Cell Signaling is the best, buy which one is good for the WB. Talk to the technical support. I have been working with phosphorylated antibodies for more than 10 yrs. and found excellent results. Please check your lysis buffer and also follow their protocols. I believe that you will get expected results. Good luck with your research.

Dear Edita,

i currently use Cell Signalling antibodies for detecting total Akt (#9272) and activated fraction (phospho-Akt ser473, #9271). I used both the antibodies for Akt detection in rat tissues (skeletal muscle, heart, brain, liver), by obtaining great specificity in Western blot analysis. I usually use for both of them a diluition of 1:1000 in 3% BSA.

Good luck with your results!

Dear Edita,

In our lab we used Santa Cruz Abs for detecting total and phospho-Akt Ser473 for WB. we have found only two immunorective bands between 60 and 50 KDa in brain tissues (hippocampus and cerebelum), so its possible that the bands you have found are the real. Good luck on your studies!

I have used #9277 (Cell Signaling, 1:300) for p-Akt immunohistochemistry on rat tissues. It worked well.

Hi Edita:

I noted that blots are from separate blots. I bet the problem is not your antibody. You should look for trivial technical solution first.

Are the tissue quality identical? Did you process the two tissues with the same lysis buffer and at the same time? Also, there are three Akt isoforms.

Solution: Run the human and rat samples on the same gel in triplicate. Probe them with a pan total Akt antibody, a rabbit-pS473 antibody and a mouse pS473 antibody.

One of the blots will give you the solution.

You don't need to buy the antibodies. You can just come over to my lab to get them.

tung

Probably due to differences in mobility between the different isoforms of Akt, especially when you get good separation via SDS-PAGE. I see the same sort of doublet using the Cell Signaling P-Akt antibodies.

I have used p-Akt Cell Signaling #9277 for rat tissues. It generated a single band.

I would propose Abcam products, such as anti-AKT1 (phospho S473) antibody [EP2109Y] (cat# ab81283)