Question (57187) | Anti-mTOR antibody [Y391] (ab32028)

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Question

I am attempting to immunoprecipitate mTOR and known interacting proteins (specifically Raptor and Rictor) with ab32028. I am successfully pulling down mTOR (as evidenced by Western blotting), but it is very inefficient and I am not seeing interacting proteins. Could I receive some advice/assistance on immunoprecipitation with this antibody? Please let me know what other info you would like from me about my protocol.

Answer

Thank you for your patience whilst I was looking in to this matter.

I checked the concentration for this lot and it is around 1.758 mg/ml. So, I don't believe it is a concentration issue.

However, I did notice that IP testing was only done on Human sample here and not mouse sample. Mouse cross-reactivity was only determined by WB only. Therefore, it could be that, although the antibody is IP'ing the mouse mTOR, the affinity of the antibody to the mouse version may be lower than that to human and that might explain why you are not seeing the co-IP of your other proteins of interest.
To help try and resolve this situation, I don’t think that any protocol tips would prove to be successful; therefore I would like to suggest that I send ab2732 as a free of charge replacement:

https://www.abcam.com/mTOR-antibody-ab2732.html



If you are interested in receiving ab2732, then please let me know, or if there is anything else that I can help you with to resolve this issue. If you could also provide either the Abcam order number or the PO# used to purchase the antibody, that would be very helpful.



I look forward to your reply.

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