Hi, I am investigating an effect of a protein on mTORC1 signaling via western blot and probing for PS6K and P4EBP1, the best known mTORC1 substrates, as well as totals S6K-4EBP1, over expressed protein tag and actin. From papers it is hard to figure how the PS6K/P4EBP1 levels are normalized. I am taking phospho over total and then normalizing for GAPDH. Should I also normalize for the overexp protein ? Please suggest if anyone knows the best way to deal with this.
Thanks
Normalize phosphorylated proteins (pS6K, p4EBP1) to their total protein counterparts (S6K, 4EBP1). Then, normalize to a loading control like GAPDH. Next, you can correlate the results with the overexpressed protein.
Kind regards
The most accurate way to normalise western blots is by using a total protein stain, such the Revert Total Protein stain, Ponceau, or AmidoBlack. Using a housekeeping protein for normalization may not be appropriate without validation, imagine that the housekeeping protein's expression may also fluctuate between different conditions.
Whereas, with the total protein stain, the only variable is the amount you load on the gel and the reason why journals recommend this method over a HKP.
This detailed information may be useful:
https://www.licorbio.com/applications/quantitative-western-blots/normalization
- Badges
- Science method