I'm interested in RyR phosphorylation state and I have specific antibodies to measure the ratio phosphoRyR/total RyR in PBMC but because I'm planning to measure it by flow cytometry I need a positive (totally phosphorylated) and negative (not phosphorylated) controls.
For cell lysates I use PKA and CIP but I don't know how to proceed in living cells.
Does CIP cross the membrane?
Is there any protocol to force protein phosphorylation and dephosphorylation in living cells available?
Any help??
Hi Pablo,
first of all, I guess, if you a going to stain the cells with antibodies against RyR, you would need to fix and permeabilize the cells anyway. So why not trying to de-/phosphorylate fixed cells with PKA/CIP before antibody staining? It might work or entirely fail, but could be a low hanging fruit if you already have everything available.
Besides this, you could try to increase total phosphorylation level in your cells by inhibiting phosphatases with ocadaic acid and sodium orthovanadate. Both worked in my hands to increase phosphorylation state of my analyzed proteins. See also here:
https://www.researchgate.net/post/Ser-Thr-phosphatase-inhibitor-for-live-cell-culture
If there is by chance an agonist which can specifically increase phospho-RyR, you could of course use it as well.
As opposite, you could use kinase inhibitor to block phosphoRyR (if known and available). If not available, I would try to inhibit some upstream kinases in signaling cascade.
Good luck!
promoting phosphorylation via cell's own PP/kinases is also a really nice approach!
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