I have incubated stromal cells with exosomes or pbs, then I detected the levels of HCK phosphorylation at Y411. I see an increase on total HCK phosphorylation (61 + 59 KDa) (p < 0.05) after SF-EVs treatment. When comparing only the 61 KDa, there is a slight increase but it is not significant p = 0.092, same happens with the 59 KDa alone.
I understood that when you measure phosphorylation after an stimulus, it is all phosphorylated or nothing. Is it possible to find different levels of phosphorylation? I am a bit confused with this result.
I have checked if there was an increase in total HCK protein being produced, and both treatment groups express similar levels.
Thanks!
Dear Helena,
yes, partial increase is possible. The phosphorylation level of an epitope depends on the balance between the kinase and phosphatase targeting that epitope. This means that the level of phosphorylation can be finely tuned by regulating both enzymes.
Normally, a western blot against the pshosphorylated and the total protein will give you the fraction of phosphorylated protein. If your treatment is not significant but you are still expecting a change, I would increase the dosage of your treatment. You could also increase the duration, but phosphorylation events tend to be very fast, so it might not change much.
Best
Hey Helena,
since you are using kDa, I guess you used Western Blot to make phosphorylation visible and quantifiable? Just keep in mind that Western Blot is not really a quantificational approach. There are some risks involved concerning saturation, background etc. To make sure, use a phosphorylation assay. You might want to use your western blot to convince your PI or boss to buy an assay. There are plenty around for HCK.
And for your question about phosphorylation all or nothing... It is basically never all or nothing. Just remember that there are many other posttranslational modifications around which might change the amount of phosphorylated proteins. And dont forget autophosphorylation.
Good luck
Depending on the number and position of phosphorylation sites, it is also possible that the pattern of phosphorylation changes upon treatment. A phospho-peptide map may show that individual sites show more dramatic changes than the total balance of phosphorylation.
Many regulatory proteins are phosphorylated on multiple sites that are individually controlled by different kinases. A 5-fold change in one site might not be obvious if other sites are already phosphorylated. The best way to look at regulated phosphorylation is to use SRM-based phosphoproteomics with mass spectrometry. In a pinch, western blots could be used if you have a good site-specific antibody available. Cell Signaling Technologies PhosphoSitePlus web site shows one for Y411 of HCK. Also, "fractional" phosphorylation only means that not all of your proteins are phosphorylated, which is the rule rather than the exception.