I working with human neutrophils. I want to develop phospho mTOR and mTOR western blot for it. Please suggest me protocol for transfer as well as antibody which is suitable for my sample.
In our Lab, we usually detect phospho mTOR and mTOR in 293 T cells and several tumor cells.
mTOR(7C10) Rabbit mAb 2983 from cell signaling
anti-mTOR (phoso S2448) antibody (ab109268) from Abcam
Tans-Blot Semi-Dry Quick reference guide From Bio-Rad. 25v, 1h.
Good luck!
I second what Hao said. I used cell signaling antibodies and they worked well for the mTOR pathway. I also used Bio-Rad and the semi-dry method as well with no issue.
Hi Durgesh,
Why would you blot phospho-mTOR ?
Aren't you interested in a more specific process related to mTOR ?
Looking at mTOR phosphorylation is too wide, you don't have the crucial information related to any process.
I recommand you read Sabatini DM bibliography (the discoverer of mTOR), you won't find any phospho-mTOR blot !
Cheers !
Dear David,
Can you please guide me how do I show 'Autophagy induced in mTOR dependent' by western blot. Do you mean I would show Phospho p70S6kinase, Ribosomal kinase?
P70S6K is mTOR's target for protein synthesis, so you won't blot it.
You should rather check ULK1 phosphorylation (inhibited autophagy), with AMPK status, since phosphorylated AMPK activates autophagy through mTORC1 inhibition. But not on mTOR ! On RAPTOR, thats why looking at phospho-mTOR is not accurate enough.
Hope it helps
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