Hi.

why did you not examine FAK (focal adesion kinase ) ? You can use the western blooting to measure changes.

https://www.researchgate.net/profile/Al_Munawir

good luck.

AL

Co-IP is very good for detecting strong/medium interaction. But for the weak or regulated interactions, such as some kinase with substrates, GPCRs with regulating proteins etc., conditions of co-ip need to be optimized. For your case, the good way, I think, is to check references and find a reasonable IP buffer, and then do some tests.

Hi Yong,

Although we never did it our-self so I would not be able to give you much detail, but you may want to consider crosslinking your proteins before IP and MS. Just as an example have a look at this paper (but you will find many others) where the authors used this strategey http://www.ncbi.nlm.nih.gov/pubmed/23613850

Hope this helps. Good luck!

Crosslinker may increase the backgroud if the condition is not optimized. So if co-ip works well without crosslinker, I don't suggest to use it.  But if you cannot co-ip those protein/substrates you are interested in, crosslinker is deserved to try.

it would be best if you can use substrate trapping mutant(s) of your kinase for overexpression in cells followed by IP. Subrate trapping mutants don't phosphorylate the substrate and keep binding to it for much longer time that wild type kinase.

the fact that you are not able to get subrtaes in in vitro pull down makes it less likely that you will get anything in IP using the same protein.

the interaction of a kinase to its substrate is short term thats why  a  substrate trapping mutant is used to check for substrates

Hi I Agree with Kunal, Find out which amino acid in active site of kinase is essential for the kinase activity, using site directed mutagenesis make a kinase dead mutant, use this mutant to pull down the interacting proteins.   

Co-immunoprecipitation is a terrible way to identify kinase-substrate interactions (with some rare exceptions). The nature of the interaction is transient - that's the entire mechanistic quality of reversible phosphorylation. Moreover, the kinase is usually present in lower concentrations that its substrates and most kinases have a slew of target proteins. In other words, if interactions were stable, signal transduction would not work very well. So, what do we do?  We epitope -tag and massively over-express a protein kinase and a putative substrate and, lo and behold, find evidence for interaction by immunoblotting. This forcing of transient interactions can be very misleading and my bet is that the majority of papers that show such interactions are doing so in a non-physiological manner.

So, what to do to identify your kinase targets?  There are many methods but one you might want to check out is called BioID in which you tag your kinase with the biotinylation domain of birA and express this in the cells of choice.  You then purify biotinylated proteins via affinity binding to Streptavidin beads and identity them by mass spectrometry. Of course, this generates false positives too and will not detect all transiently interacting proteins, but it's a lot better than co-precipitation and has the enormous benefit of pushing you away from biases by looking under the lamp post.

There are a lot of recent studies of the approach, you could start here: https://www.researchgate.net/publication/260131454_BioID_A_Screen_for_Protein-Protein_Interactions

Article BioID: A Screen for Protein-Protein Interactions