As an alternative to in vitro phosphorylation, you can mutate the suspected aminoacid to D/E to produce a phosphomimic mutant. The structure of D/E resembles the structure of pS/ pT.

There has to be plenty of literature on this. Depending on the residue that needs phosphorylated, you need to add, next to ATP, the appropriate protein kinase.

As an alternative to in vitro phosphorylation, you can mutate the suspected aminoacid to D/E to produce a phosphomimic mutant. The structure of D/E resembles the structure of pS/ pT.

First try to define in which physiological conditions your complex could get phosphorylated in vivo. This could help you to narrow down the number of potential kinases to try in vitro. Alternatively you could also search phosphoproteomics databases to see if phosphorylation sites are already been described.

Good luck.

Hi, Thierry. Please try to perform our Phos-tag SDS-PAGE procedure. This affinity electrophoresis enables the mobility shift detection of phosphorylated protein from the nonphosphorylated counterpart. And also, it enables the label-free detection of all phosphoprotein species regardless of the kind of amino acid residue. Of course, the radioactivity is avoided. I attach our article, which demonstrated in vitro phosphorylation of recombinant proteins by using Phos-tag SDS-PAGE.

...is your protein a kinase itself? If so then auto-phosphorylation can be carried out by simply incubating your purified protein with ATP (varied conc - 50uM and upwards, with 5-10mM MgCl2 or MnCl2) for 30 minutes, 37 degrees. Dialyse out the ATP afterwards (if necessary). In parallel, incubate the same protein with Lambda-phosphatase to ensure no phosphate groups are present (unless you are sure that it is not phosphorylated in the first place). If it's not a kinase then, as suggested above, you'll need to get hold of (or clone and purify) the appropriate kinase - not a 'simple' option there though.

Hello Thierry, I agree with the comments above, particularly those of Eiji Kinoshita and if you haven't done so already, suggest the kinase site prediction algorithms hosted at the Danish Technical University: http://www.cbs.dtu.dk/services/NetPhosK/ to narrow down your search. Also, we have found that purified recombinant proteins can be phosphorylated by recombinant kinases that don't actually phosphorylate those particular sites in vivo - this may work to your advantage!

Check the sequence of your protein in PHOSIDA database and you'll find all the sites that could be phosphorylated and the kinase you'll need to do so. You can find many kits for in vitro phosphorylation, the conditions of reaction can vary for one company to another, but they are more or less the same. I would recommend you the one kit from Sigma/aldrich or invitrogene. Please check the buffer reaction composition out. Once I had some problems with protein stability due to that!

and, I definately agree Didier's answer!!

good luck!

The basic components needed for in vitro phosphorylation study are (1) kinase, (2) substrate, (3) Mg-ATP in some appropriate buffer. Please set up your study according this basic idea.

In your case, you seem to have substrate as a recombinant protein. How to obtain kinase would be a key point in your study. A dirty, rough experiment would be to use crude cell homogenates.

How to detect/analyze phosphorylation ? You may use p32-ATP or phos-tag SDS-PAGE as Dr. Kinoshita suggested. Phos-tag SDS-PAGE does not require radioisotope.

I hope these will be useful.

The first thing to consider is how to phosphorylate your protein of interest. If it is an active kinase then auto-phosphorylation would be the preferred route. If it is a not, then is it a known substrate for a kinase you could acquire? You could also try doing phosphorylation using a commercially available kinase from the same family with a known upstream kinase of your protein. Once phosphorylation is achieved, the next stage is to compare the binding properties between before and after phosphorylation. You could use pull-down assay if one of the binding partner is affinity tagged, for example: GST tag or HIS tag. There are other biosensor based assays you could also consider if more detailed answer about binding properties is required. You could also start mapping the phosphorylation states of your protein in parallel with the biophysical characterization. This is especially important when there are multiple sites of phosphorylation.

Thank you to the all of you; this helps.

According to the elements suggested above I would first run http://www.cbs.dtu.dk/services/NetPhosK/ or/and PHOSIDA. We have some ideas of the possible involved sites on the protein and could probably restrict potential kinase(s?) to purchase (I guess commercialy available kinase are expensive and we don't want to start a cloning expression procedure of these babies).

Mass spec and Eij Phos-tag SDS-PAGE to see if the protein pattern is modified after kinase treatment. We would then perform our specific fluorescent binding test to measure Kd and compare.

Any more suggestions and remarks are of course welcome.

It will probably be difficult to get quantitative phosphorylation no matter what your conditions are by an in vitro method. So either you are going to have to purify the phosphorylated protein, which may have different isoforms that will copurify, or design an assay that deals with a mixture, which sounds easier in your case. BTW, if you are going to do this in vitro, an alternative to recombinant kinases is old-school purification from primary tissue. Heart-muscle kinase for example is very easy to purify and one cow heart gives you millions of dollars of commercially purified enzyme.

Alternatively, you can try to express your protein in a eukaryotic system, such as yeast or baculovirus. You may or may not get phosphorylated forms, which may or may not be in the right places and overall expression levels will be lower than for Coli. But depending on your setup, it may be fairly easy to try.

If you are going for the mutagenesis route, a more conservative loss of function mutant (change to non-phosphorylatable form) is likely going to be more informative than trying to mimic the phosphorylation with an acidic residue though that sometimes works, and of course you can try both.

Sounds interesting - good luck.

Hi Thierry

I think you got almost all answers to keep you busy for some time. However there is one more.

You have both proteins purified, right.

And you suspect that one of them could be phosphorylated. If that protein is mammalian there is an easy way to phosphorylate the protein in viro using cold ATP by incubating your protein 5-20 minutes at 37C with a crude lysate (cells lysed in mild buffer contining 1%Triton) activated 15minutes with staurosporine or Pervanadate (orthovanadate+H2O2) 1.

Then, you can compare the binding of your proteins performing a simple pull down, using as control the "non phosphorylated" (i.e. incubated with the lysate w/o ATP).

The ammount of lysate varies, but 100-200 micrograms should be enough. The ammount of the recombinant protein for a pull down could be 1-2 mcrg.

We performed some similar assays long time ago(search for Torres and Pulido), when hot ATP was still in fashion.

This approach involves the phosphorylation of your protein by many different kinases but gives you a hint about the role of the phosphorylation in your assays.

Hi Thierry,

These are all great ideas and a quick and dirty experiment will give you a hint at the interaction interface between two proteins. However, a negative result (as well as a positive result) will be difficult to interpret since it will be hard to know that your that your phosphorylated protein is indeed phosphorylated on the putative binding region unless you do mass spec or 2D gel analysis. Random phosphorylation with a random kinase may give aberrant results. An alternative would be to make a phospho peptide (or a native peptide that you can phosphorylate yourself in vitro) surrounding the binding region and do a simple pull down assay after incubation with your recombinant protein. You can detect binding differences between non-phospho and phospho peptide and get relative binding affinities. Keep in mind that this is not in the context of the entire protein which could affect binding affinity either positively or negatively. Phospho-memetic point mutations in the full-length protein will be required to truly identify the effects of phosphorylation on binding. The most informative experiment would be in-vitro phosporylation of both wt and alanine point mutants and measure association by co-IP. No binding with the alanine mutant would identify the critical phospho sites while confirming that there are no other phospho sites involved in the interacting motif. Good luck!

Hi Thierry,

to identify the physiological upstream kinase, you may profit from our KinaseFinder service which is further described on ProQinase's website: http://www.proqinase.com/content/view/90. Using a sample of your purified protein (or a peptide sequence instead) we may be able to identify kinases which are able to phosphorylate your protein.

Hi Josema,

 Can you send me your cold in vitro recombinant expressed protein phosphorylation method?

Here is my e-mail address [email protected]

many thanks

Baki

Sorry A. Baki,

did not read your question until now.

You can find it here: http://jcb.rupress.org/content/147/6/1129.full.pdf+html

Let me know if you have any other question regarding this assay ([email protected])

Now some engineered E. coli strains are available to insert phospho-Ser in desired places: https://www.addgene.org/rinehart/