I would like to know what may be happening in my Western Blot where I didn't see changes in the phosphoprotein (control and stress conditions), but the total protein has disappeared in the stress condition while remain unchanged in the control condition. Also I saw mild changes in my endogenous control during the stress conditions (observed previously). I read about activation of proteasome by phosphorylation (activation) and other possibilities. The protein is eiF2a and peiF2a.
It sounds like you have loaded less protein in the "stress condition" lane. Did you ponceau stain the nitrocellulose membrane? Is your "endogenous control" also reduced in the "stress condition" lane? What is your stress condition? Thapsigargin treatment works very well as a positive control for functional p-eIF2a antibodies.
I agree with mark. In my view, you are seeing equal phosphoproteins because of low abundance and saturated antibody binding in all samples while as variability is reflected in total protein because of high abundance. How did the loading control look? If this is the case, there should be difference in loading control. Second, did you use phosphatase inhibitor in your protein extraction cocktail?
I´m afraid is not a problem of load. Because I have reviewed and reloaded again, and the WB is absolutely the same. The total protein amost disappeared. My stress condition is Hypoxia. I think first I need the Thapsigargin control, but I remain intrigued by which mechanisms the total protein goes while the phospho is the same and the meaning of it.
And anseweing the second comment, small differences in load control can not explain the disappearance of the signal of the total protein. And my extraction cocktail contain phosphatase inhibitors and protease inhibitors too.
Thank you very much for your answers.
You can see my WB here.
Bibiana, I have one interpretation. Phosphorylation of the eukaryotic initiation factor 2 (eIF2) α subunit is a well-documented mechanism to downregulate protein synthesis under a variety of stress conditions. Hence this result could be due to increased production/stability of the eIF2a phosphoprotein under stress to stop translation and survival. In the meanwhile due to stress the cell stopped translation and production of factors like eiF2a involved in translation. read this articles and sorry if i am incorrect.
http://www.ncbi.nlm.nih.gov/pubmed/10216940
http://www.ncbi.nlm.nih.gov/pubmed/11575161
So to generalize, there are 3 possibilities:
1) Technical problems, including the antibody not working correctly.
2) The biology not cooperating: eIF2a is not phosphorylated in response to hypoxia in your samples
3) Variability/Noise….You are studying a process with a certain mean and standard deviation and you happened to isolate an outlier.
Mark, seems as if you misread the problem. She is getting the phospho but there is a rapid reduction of total protein. Then how can you say that eIF2a is not phosphorylated in response to hypoxia? Please see the blot picture she provided in the discussion. Sorry if I am the one misread it :)
Thanks Sivadas…I hadn't seen the uploaded results. I think the generalizations still hold, however….that's the beauty of generalizations. Here are some specific examples for each:
1) The total eIF2a antibody might not be a total eIF2a antibody…maybe it is recognizing a different protein or its detection of eIF2a is being blocked by some post-translational event (phosphorylation, cleavage, etc.)
2) It seems like eIF2a is not really being phosphorylated in the system in response to hypoxia. The high basal level of eIF2a is a bit strange…again, it should be confirmed that the phospho-eIF2a antibody is working correctly.
3) The 3 control lanes and 5 stress lanes is convincing that this is not an issue of variability. The light background on the stress lanes side of the blot is a bit concerning, but I agree…this probably isn't experimental noise.
But focus on making sure the antibodies work….when I developed this one 14 years ago, I was very happy to figure out that Thapsigargin treatment was such a good positive control:
http://www.cellsignal.com/products/images/9721_fig2.jpg
Check your antibody data sheet or with the manufacturer - maybe your total eIF2a is binding exclusively to non-phosphorylated protein. Thus, as suggested above, if no new protein is translated while all eIF2a present is being phosphorylated due to your stress condition the "total eIF2a" AB may find no place to bind.
Also other are other post translational modifications that may block binding of the AB to eIF2a - check the antigen it was raised against.
Good luck!
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