I wonder Rohit whether you want to "find" the kinase that phosphorylates your protein or any kinase that may phosphorylate your protein. Please note that kinases may have more than one  endogenous substrate.

I'm interested in finding the kinase that phosphorylates my protein (eIF4E) under  specific starvation conditions.

The easiest way is with Ip/CoIp by Immunoprecipitation of eIF4E and followed with western blot with a panel of kinases: hopefully you will find some candidate according to the literatures /publications. Such as

mTor

MAPK and  (MNK1 & MNK2)

CDKs

 The other is more expensive method is you fish out all proteins which are in complex with your eIF4E and hopefully you will find a kinase(s) :

You immunoprecipitate eIF4E and then take you’re a.b-ag complex and run LC-MS/MS to identify the proteins under the conditions your cells have (starvation or inducement).

In this experiment I suggest you use a purified antibodies and also have a.b as a negative control to exclude the proteins, which are in serum.

Good Luck

Thank you Fuad Bahram for the answer . 

But don't you think that interaction between kinase and substrate is transient and it is difficult to find a kinase in Ip?  I have tried doing pull down but the only kinase that we pulled down and identified by mass spec was pyruvate kinase.

Personally I don’t have any experience of eIF4E studies but of Ser-phospho kinase identification. And certainly will Pyruvate kinase be included in MS / MS identification because there is a lot of and easy to find when you have total protein extract in MS / MS analysis, but to exclude proteins that are not in the complex should immunoprecipitation done first and then the MS / MS analysis. The result should be then analysed and exclude proteins, which are note of interest.

In some cases enrichment phospho-protein are necessary such as: through accumulate phosphorylated eIF4E (starvation time? ) or by cell cycle analysis, or inducement.. then Ip..followed by LC-MS/MS.

Rohit, the kinase that phosphorylates eIF4E is Mnk, a MAPK-interacting serine/threonine kinase.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2953056/

Thank you Andre Fernandes for your reply.

It could be that 4E that I'm working with is also phosphorylated by Mnk but I have got a different phosphorylated serine and our model system is different. So I have to experimently identify and establish the role of kinase.

Since we have not yet published our results so I would not like to give out exact details of my results,.

Thank you Fuad Bahram for answer.

I have tried pull down which is identical to immunoprecipitation and analysed by mass spec but mass spec failed to pick any relevant kinase. 

I could try phospho enrichment to remove non-phospho proteins and lets hope to find a kinase.

Just as a byline: If you do IP/CoIP you have to know where your AB binds to your target protein. Different ABS may pull down different fractions of your protein. E.g. an AB that binds to the prot./prot. interaction site will only pull down the fraction that is currently NOT bound to your target. So it may be interesting to check your ABs antigen and/or try different ABs for the IP.

Good luck!

well, i don't have any experiences in kinase identification. I think if we know the previuosly mechanism first, we can predict easier.