What is the molecular weight of mTOR expressed through pcDNA3/AU1-plasmid containing rat mTOR gene. I am using this plasmid and when I immunoprecipitate myc tagged mTOR with myc antibodies, I am getting a band at around 100 kDa mark. The m.wt of human mTOR is around 250 kDa.
Hi,
Do you try only western or RT-qPCR for mTOR before IP? It would need to check mTOR expression in cell. If expression of mTOR is low or not expressed, you might need to check plasmid or transfection again.
Is it possible that mTOR gene is not full length in the plasmid? If it is full lehgth in plasmid, the plasmid will too large to transfect, and may need try transfection condition.
Are you certain there is a myc tag? I thought this had only an AU1 tag. We always had much better luck with the human FLAG-mTOR constructs to see a band by western.
Hi.....that might be a non-specific band. You should check it in your input lanes. You should get it at 250. I agree, u should check first the expression of the gene by western blot. You might need to check transfection as well as your immunoprecipitation procedure.
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