I'm trying to analyze how the ratio of AMPKp: AMPK changes among my treatment groups. What is the best way to do this? Currently, all I've done is divide AMPKp by AMPK, and then calculate differences by diving average ratio of experimental by control. However, these results completely go against many published literature articles.
Is the best way to do this by normalizing with a loading control such as actin first, and then divide the ratios?
If I understand you correctly, you have measured how many percent of your AMPK are phosphorylated. That is one part of the story, the other would be possible changes in total [AMPK].
Did you measure the quantity of AMPK and AMPKp on the same gel? If so, you just have to divide AMPKp by AMPK for each well.
If you do it on different gels, you should normalize the quantity using a protein that is not modified by your condition (ß-actin, ...). Then you could divide AMPKp by AMPK to obtain your ratio.
Phosphorylation of AMPK has to be determined as ration pAMPK/total AMPK. This ratio is your result and shows influence of any stimulus on AMPK phosphorylation in your experimental setting.
Additions normation with any "housekeeper" expression/determination or any other control will give you no further information in this regard. The only answer this would deliver could be whether there are loading discrepancies or other methodological sources of uncertainty/mistakes often based on the experimentator and working method but not related to the experimental setting /culture/organism.
Additionally, as also mentioned by Frederic, this should be done in a comparable manner (maybe on the same gel).
Alternatively you could perform the same analysis using adequate ELISA assays, leading to quantitative results (if this is a goal). However, semiquantitative analysis via WB and results on the ratio phospho/total AMPK should be sufficient!
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