I need to study the effect of the protein Y mutation on the levels of a protein X. So I over-expressed the protein X under 5 conditions:
(1) not over-expression of protein Y
(2) with over-expression of a wild type protein Y
(3, 4, 5) with over-expression of Y mutants
The over-expression of the Y mutants is not the same due to the mutation. Because of these differences probably I should normalise the X protein level with tubulin but also with the normalised levels of the mutants?
How can I compare the X protein levels to get some information about the effect of Y mutation on X protein level?
I've not done this sort of experiment previously, but I imagine that you can run all five samples on the same gel and develop bands for tubulin (or whichever loading control/housekeeping you use) and for Protein X. Then for analysis you can normalise first to tubulin and then normalise these values to the tubulin-normalised lane 1. This is similar to what we do following untreated/treated with drugs etc. I assume you are using ImageJ or something similar to quantify blots? I have attached a quick Excel file which shows an example of the calculation using made-up values. Hope this helps.
It would likely be worth developing bands for Protein Y if you have the antibody for it, first to confirm the overexpression of the wild-type, and also potentially the mutants depending on if the antibody for Y can recognise mutant protein.
Not entirely certain this suggestion is appropriate, but hope it helps. Good luck with your research!
Thank you Emyr for your reply and the useful excel file. I have still a question: you think that I should also compare the protein X levels to the wild type and mutants Y protein. What bother me it is that the protein Y mutants express much better that the wt so I had to adjust the amount of DNA for the transfection. So I don't know if I should consider this difference in my calculations.
Hi Mara. I'm not certain how to proceed with normalising to Protein Y levels. I think it would depend on the amount of Protein Y (WT or mutant) that is observed after transfection. Even if different amounts are transfected, I assume that what is important is the observed/functional protein at the end of it. So different results will be obtained if you normalise to the amount of transfected DNA or if you normalise to the amount of observed protein, if differences in translation are seen. I think normalising to the observed protein makes more sense since that is generally what is functional.
So if you have an antibody for Protein Y, I think it would be worth developing bands for Protein Y (WT+Mutant, assuming the antibody detects both). Then in the analysis you can add an additional normalisation step for Protein Y, though I'm not sure of the best way to do this. If anyone has any additional advice that would be useful.
Good luck!
Best,
Emyr.
Thank you Emyr! I presented my results normalising protein X to tubulin and then to protein Y and it seems that the analysis made sense.
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