I want to check whether my protein is undergoing in vitro phosphorylation by substrate which is a Kinase. I am working on plant proteins. I am planning to do the reaction as follows,
MBP-Protein (X)
GST-Kinase (Substrate)
ATP
1X PBS
Reaction at 30C for 1 hr. detection by Anti-MBP.
Can anyone who has experience of doing In vitro Phosphorylation of Plant proteins kindly share with me?
Note: I checked the phosphorylaiton site by in silico (NetPhos 2.0).
Dear Kesava,
Before going through the complex phosphorylation assay involving kinases I would suggest you check for phosphorylation first in the western blot if the phospho-specific primary antibody to the target protein is available. You can compare the increased or changed phosphorylation status by running suitable controls for proper comparison.
When you treat cells in vitro and prepare lysates, don't forget to add phosphatase and protease inhibitor cocktails to catch the phosphorylation in the WB.
If phosphospecific antibodies are not available still it can be detected by modifying the experimental approach. Most phosphorylation happens to Tyr or Ser residues. Yo can also IP with pTyr or pSer antibody (s) and run the material for WB and probe for total protein. That will also recognize a selective pull of phosphorylated protein in a given experimental condition.
Substrates are usually defined relatively depending on the enzymes that work on them and the same enzymes can also be substrate for some other enzymes for different biochemical pathways. So it is quiet possible to see the phosphorylation.
Hope that helps.
Dear Kesavan,
in addition to the excellent advise from Amritlal some things you should consider when planning in-vitro kinase assays: PBS is not a good choice for kinase assays as high phosphate levels may inhibit kinases, better use TRIS or HEPES as buffer salt. You did also not mention at which concentration you are going to use the ATP, with the approach you are considering 1-5 mM would be advisable. And finally don't overestimate the significance of in-silico predictions for the determination which kinase is actually phosphorylating a protein of interest at a specific site in-vitro.
But basically if you see an increase in western signal strength after you have incubated MBP with your kinase it is a good indication that it is a target for this kinase.
A few modifications and caveats. Not all GST-fusions of kinases are active. Better, if possible, to access purified, soluble proteins (these can be expensive but send around an email to others in your research facility). Using two bacterially derived proteins is very artificial as they are at supra physiological concentrations. Kinases are usually present at much lower amounts than their substrates. I suspect that you will get a positive result due to the NetPhos prediction but that will not be necessarily meaningful as many kinases share primary sequence requirements around phosphorylation sites. It is essential to show that the kinase you selected is relevant to your protein X in vivo. In plants, this is tricky since you either need to suppress the kinase genetically, or with a small molecule inhibitor. Kinases in vitro are far more promiscuous than in cells. Finally, not sure what you mean by detection by anti-MBP. Just use gamma-32P-ATP and measure incorporation of phosphate directly (either by precipitation on P81 paper in phosphoric acid, or run on a slab gel. If you expect to see a gel shift, you may be disappointed. Don't be afraid of radioactivity, The amounts needed are minuscule (micro-Curies) and this is much more sensitive than other assays. If you don't have a radiation license in the lab, then find one that does and arrange to go there.
Longer term, it sounds like you know the site. Generate phosphospecific antibodies and evaluate the conditions of phosphorylation of your protein under various conditions (stress, etc). These should coincide with the known regulation/agonists of your putative kinase. If not, you've likely got the wrong one....
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