I want to study PI3K/Akt Signaling pathway in human and mouse samples using Western Blot. Does anyone know antibodies for Akt signaling pathway intermediates which works great in Western Blot, especially for phospho-antibodies?
Thanks in advance
Antibodies we use:
PTEN - Santa Cruz #7974 , mouse, 1:1000 dilution (56 kDa band)
AKT - Cell Signaling #9272, Rbt, 1:2000 dilution (58 kDa Band)
P-AKT - Cell Signaling #9271S, Rbt, 1:1000 dilution. (58 kDa band)
These Ab work very well with western blot for human, mouse, and canine proteins. Overnight (16hr) incubation.
Which other proteins are you looking at?
I used both PTEN and AKT ( p and total) from cell signaling technology and worked great for me.
Fo AKT and mTOR, we use cell signaling tech and for IRS2 we use Millipore
Other Antibodies would be;
Phospho-4E-BP1
Phospho-S6 Ribosomal Protein
and S6 Ribosomal Protein
Thanl you for your all answers, but if it is possible could you please suggest Abs with their cat. no. And, could you also please share your optimized protocols for these specific phospho-antibodies (p-AKT, p-mTOR, etc.).
Thank you,
We use Cell Signalling sampler kit 9916 Phospho-Akt Pathway Antibody Sampler Kit. It gives good results and is enough for 10 blots at least.
It is depends of your model (animal, cells, host of the antibody). Taking into consideration all this look for works with a similar model and a closer intervention to chose your ones. It is the smart option always. I attached you a webside with many references of each proteins that you are looking for. You have there publications with different proteins and different points of phosphorylations. All that the people say here is okey. However to AKT always recommend include Thr308 because both (Ser473 & Thr308) are needed for a full activation of AKT.
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