In my experiment using C3H10T1/2 cells I found phosphorylation of PKA substrates at band size of In-between 75-100 and 100-150 and also at 150 position. What might be those target proteins?
I used CST antibody 'Phospho-PKA Substrate (RRXS*/T*) (100G7E) Rabbit mAb #9624'
Hi, It will be very hard to predict the identity of the bands as there are too many possibilities. In addition, the bands coming up with the PKA-substrate antibody is in no way conclusive proof that they are being phosphorylated by PKA. There are many kinases which can phosphorylate at PKA-like motifs. I am not sure about your objective, but to verify that these are indeed PKA targets in your cell system, you may use some inhibitors (or activators like Forskolin, if the system permits). The predicted targets and known substrates can be found at -
http://phosphonetworks.org/nameCheck.pl?id=PKACA
http://phosphonetworks.org/nameCheck.pl?id=PKACB
http://www.kinasource.co.uk/Database/substrateList.php
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448854/
If you really want to identify the phospho-proteins, you may have to some way fractionate the lysates (eg. size, substrate-motif antibody IP), combined with inhibitor controls and go for MS analysis after phospho-peptide enrichment.
Good Luck with your analysis.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes