I am unsure of the cause. It is reproducible across multiple blots and cells lines with both the phospho-specific antibody and the total mTOR antibody. Thanks for the help!
There are two main most likely possible reasons for your observation:
1) A fraction of your protein is glycosylated (or any other post-translational modification) and it might have increased it's molecular weight.
2) The lower MW fragment of your protein is a product of proteolytic degradation (you might want to include protease inhibitors at the stage of the cells lysis / extraction). Very weak 3rd lower MW band might confirm this assumption.
Thanks for your answer! I'll look into the role of protease inhibitors. Some were already included in the lysis buffer, but it's worth investigating.
Yes, it definitely could be post-translational modification. One way to test is to treat the sample with either phosphatase or glycosylase, and see if it collapses down to a single band.
I have seen multiply phosphorylated forms of target protein run VERY differently from one another (see Fig 7 in attached paper).
Recently, I heard a talk that described a protein with a different apparent MW from what was known and (repeatedly) published. Turns out there was an in-frame methionine that generated a smaller fragment, but the existence of a larger form was unknown. Something to consider.
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I agree with the previous suggestions, focus your attention to the prevalent band. The lower band could be a degradation product, and however it is weaker than the first. I assume that the higher is mTOR protein.
You may want to add a phosphatase inhibitor to your lysis buffer as well. It depends on the inhibitors you are using, but if you are looking for total protein, the recommended amount of protease inhibitors plus half of the recommended phosphatase inhibitor cocktail usually gives you clean bands.
I use the Cell signaling antibody for mTor on several human cancer cells and have not seen these double bands.
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