You have at least couple options:

https://worldwide.promega.com/products/cell-signaling/kinase-assays/peptag-non_radioactive-protein-kinase-assays/

http://www.abcam.com/universal-kinase-assay-kit-fluorometric-ab138879.html

If you are referring to a protein kinase, there are numerous commercially available options, thanks to the interest of the pharmaceutical industry in kinase inhibitor drug discovery. There are peptide substrates for many kinases, fluorescence-based catalytic assays and ligand binding assays, and various ADP detection methods. The choice depends on what type of equipment you have available, how much you can afford to spend, how much throughput you need, and what kinase you are working with.

Sir, i'm interested in sphingosine kinase and because of lack of proper funding we cant afford too much in kinase assay. I'm looking for relatively cheap protocol for kinase assay /

If you have well-purified enzyme to work with, you can measure ADP production. A continuous spectrophotometric assay for ADP production for which all the components are available at relatively low cost is the pyruvate kinase/lactic dehydrogenase system. A bonus is that it regenerates the ATP so you don't have to worry about ATP depletion

ADP + phosphoenolpyruvate pyruvate + ATP (pyruvate kinase)

pyruvate + NADH lactate + NAD+ (lactic dehydrogenase)

You can buy the mixture of coupling enzymes as a stock solution, phosphoenolpyrivate solid, and NADH solid from Sigma.

You have to work out the amount of coupling enzyme mixture to use under your conditions. You have to use a high enough concentration so that the coupler enzyme is not rate limiting (typically about 10 U/ml of pyruvate kinase is sufficient). I normally use 0.5 mM NADH and 1 mM phosphoenolpyruvate. Solutions of these should be prepared fresh each day from solid.

The reaction is monitored by the decrease in absorbance of NADH at 340 nm. The extinction coefficient change is 6.2 mM-1cm-1.

You need to add Mg2+, ATP, sphingosine or other substrate, and the sphingosine kinase enzyme. To measure the background rate of ATP hydrolysis, leave out the sphingosine. Start the reactions by adding the sphingosine kinase, leaving time beforehand for any ADP in your ATP to be converted to ATP by the coupling system.

sorry im not working on purified enzyme, im working on cell lines. I wanted to know kinase activity in a cell under given condition. So far i have come across use of NBD-sphingosine (http://www.sciencedirect.com/science/article/pii/S0003269703008285) for sphingosine kinase assay

Mohd, attached is a protocol for measuring SphK based on the use of radioactive ATP.  

Initially you can go for single conc inhibition assay process to check whether it is active or not?

(www.kinase-screen.mrc.ac.uk)

go on this web site , you will find cheapest price for this purpose.

best of luck

First of all, I would like to apologize if this is not the proper way to make a question (maybe because this process is regulated by a ResearchGate forum regulation that I do not know). But I would ask to Dr. Shapiro, if I may, if you had any reference to the coupling enzyme assay you are suggesting (to know the conditions and kinetics for the coupled enzymatic reactions). I am trying to measure the SnRK1 kinase activity from plant desalted extracts, but I lack the installations to measure this activity using the traditional radioactive protocol. I have a protocol with specific reaction conditions that includes the substrate for this kinase, so I hope it can be used as an argument to justify the use a coupling enzyme photometric assay.

Best wishes for everybody in this conversation.

Ismael,

The premise of coupled enzyme assays for kinases is detection of the ADP product. For this to be practical, it is important that the level of hydrolysis of ATP to ADP by other enzymes in the sample, plus the level of spontaneous ATP hydrolysis, is low compared to the activity of the specific kinase of interest. This can often be achieved when working with highly purified enzymes, but may not be achievable when working with crude extracts. In such a case, it is better to measure the phosphorylation of the AMARA peptide substrate. Since you lack the facilities for radioactivity work, you can consider 2 options: purify the SnRK1 kinase to a sufficient level to enable the use of ADP detection, or use a non-radiometric method for detecting phosphorylation of the peptide.

There are several nonradiometric ways to measure peptide phosphorylation. The way you choose will depend on what facilities are available. Do you have phospho-AMARA-specific antibody that would allow you to use an immunological approach, such as ELISA?

The peptide is probably too small for SDS-PAGE (Mol. wt. about 1500), although it might just be in range of Tris-Tricine gels. Phosphorylation would probably change its rate of migration on SDS-PAGE.

Do you have an HPLC or capillary electrophoresis apparatus that could be used for peptide separation? If you have the peptide synthesized with a fluorescent dye attached, and you have a way of separating phosphorylated from unphosphorylated peptide, and a way of detecting and measuring the fluorescence, then you can follow the phosphorylation reaction by following the fluorescent phosphopeptide, even when using a crude extract.

Thank you for your reply Dr. Shapiro. I will explore your suggestions for measuring SnRK1 activity, starting with a way to purify the SnRK1 kinase from my extracts or considering an immunological approach.

Adam B Shapiro glad to see your various answers posted here (though I'm quite late to the party, as this thread is 3 years old). I'm trying to adapt a protein kinase project to be feasible by undergraduates (i.e. minimal expertise) in an environment with minimal infrastructure (i.e. filter-based visible microplate reader, luckily with a 340 nm filter).

I'm accustomed to this methodological principle, as I've previously used it to assay metabolic kinases (e.g. creatine kinase in the creatine + ATP --> phosphocreatine + ADP direction) but have not as yet attempted it with protein kinases. I'm wondering why I'm having a devil of a time finding more references utilizing this principle -- as opposed to radioisotopes, fluorophores, antibodies, etc.. Certainly, there are the difficulties you've pointed out; purity of preparation, rate of specific vs nonspecific ATP hydrolysis, and so forth. Still, I'm surprised that when purified kinases are available, this isn't a more prevalent method.

I think the reason is that absorbance-based methods are not sensitive enough for protein kinase assays.

A protein kinase has 2 substrates: ATP and a protein or peptide that gets phosphorylated. The extinction coefficient decrease of NAD(P)H -> NAD(P)+ is about 6200 M-1cm-1 (=0.0062 µM-1cm-1) at 340 nm. In a microplate, the path-length through the sample in a pretty full well is perhaps 0.6 cm, depending on the volume. A decent absorbance change is at least 0.1. From this information, you can calculate what concentration of product must be formed, which is (0.1)/(0.6)/(0.0062)=27 µM. For measuring an initial rate, the product formed should be less than 10% of the substrate concentration. Therefore, the substrate concentration has to be 270 µM. This is not problem for the ATP, but it gets expensive when using a synthetic peptide, and is very unlikely to be practical for a whole protein. You can use a 384-well plate to minimize reaction volume, and thereby the cost of running the assay, compared with a 96-well plate.

A more sensitive absorbance-based method is to detect the phosphate product in endpoint mode using ammonium molybdate/Malachite Green reagent. (Edit: For this to work, you would need a phosphatase to dephosphorylate the substrate as fast as it was phosphorylated.) With that, you can get a decent absorbance signal with about 5 µM product. Unfortunately, it is not possible to use this acidic reagent in continuous mode, like the above-mentioned continuous assay.

Fluorescence-based assay methods are way more sensitive than absorbance-based methods.

https://www.jenabioscience.com/nucleotides-nucleosides/nucleotides-by-application/on-proteins-enzymes/kinase-signaling/non-radioactive-protein-phosphorylation-analysis/nu-833-590-gamma-6-aminohexyl-atp-atto-590

Hi Mohd. Arish

did you ever try PamGene's kinome activity array platform that can measure functional kinase activity of >320 kinases in a multiplex manner followed by bioinformatic analysis +downstream cellular signaling?

https://pamgene.com/technology/

Hi guys,

Please can anyone tell me if there is a on-specific kinase that can be used to phosphorylate a protein? We are investigating the activity of a kinase but the signal we are getting is very low, so we have some suggestions that maybe the kinase requires phosphorylation too before it can be able to phosphorylate it substrate effectively. So I want to know if there is a non specific kinase that can be used to achieve that purpose.