Hello Huimin yap, I'm not specifically involved in mTOR IP, but there is a general rule valid for IPs: the antibody you use to IP must work in IP, and that is always the critical point. It means the Ab has to be raised against an epitope that is available and recognizable in a native state of your target protein. I could deduce that the reason why you always see IP with mTOR and immunoblotting with Rictor is that the mTOR Abs work well in IP, while the Rictor Abs do not, or not so well. But it is also possible that people just follow already tested protocols because they risk less, since it is known to work already, so you can still try to do the opposite and see if it works.

Another possibility resides in the balance between the complex and the free components. As I said I'm not that much into the mTORC2 complex, but if all mTOR is incorporated in the complex while only 50% of Rictor is, IPing mTOR you maximize the yield of Rictor, while if you IP Rictor, statistically only 50% of what you IP will be bound to mTOR, so the yield in the co-IPed mTOR would be reduced. I'm hypothesizing possibilities, this you might know better from what is known about the complex!

Last point, concerning the IP technical aspects. One possibility is to bind the Ab directly to sepharose beads, as you seem to imply you are planning to do. But another possibility is to incubate the antibody with Protein A or G (depending on the type of Ab, as a rule of thumb rabbit IgGs or mouse IgGs, respectively) which very strongly bind to the invariant region, mostly the Fc region, of IgGs and then use these beads with the bound Ab in the IP protocol (you can also do it the other way round, incubate the Ab with your extract and then pull-down the Ab that will be bound to the complex with Protein A or G).

Finally, you actually mention mTOR conjugation to sepharose beads, not the Ab's conjugation. If this is your plan, you are talking of a pull-down, which is a bit different. This means producing recombinant mTOR or Rictor and binding it to sepharose beads. When you incubate this with the cell or tissue extract you will induce an exchange of the physiologically incorporated mTOR or Rictor with the exogenous you are providing in great excess, so the exchange is based on a mass effect and then it should be pretty much the same thing if you use mTOR or Rictor!

Good luck.

Thanks Dr. Pietro Pilo Boyl.