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Kinase Assays: Specificity And Speed
Buying Tips
Dec 19 '06
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Introduction |
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| Just as there are many types of kinases in the “kinome” – the total collection of functional kinases in an organism’s living cells – so are there many kinds of kinase assays that researchers use to study them. If you are a novice when it comes to measuring the ability of your kinase of interest to phosphorylate a substrate (or the degree to which your substrate of interest acquires a phosphate group due to the phosphorylation activity of a kinase), the variety of kinase assays from which to choose can be befuddling.
As for choosing assay type, Annegret Boge, director for reagent and assay R&D at Molecular Devices, advises considering the ultimate assay goal: “I would recommend looking very carefully at the final goal for the assay. For example, does the customer want to run one kinase or many kinases? Is the throughput just a couple of plates a day, or will an [ultra high-throughput] screen be the final application? It is amazing how small things like an extra pipetting step or a wash step, which seem to be easy at low plate volumes, becomes very labor intensive when many plates are run.”
Another perspective on choosing an assay type comes from Michael Curtin, product manager for in vitro assays at Promega. “Choosing the right assay for your needs depends largely on the source of the kinase activity and the desired throughput,” he explains. “Numerous assay technologies are available; some are best suited for use with purified protein kinases, while others allow the researcher to measure the activity of a particular kinase in a crude tissue or cell extract.” Your kinase activity may be found within protein mixtures in cell extracts, or “highly purified recombinant protein kinases expressed for the purposes of high-throughput screening applications.”
The types of lab equipment that are available to you may also play a role in which assay you choose. “If the lab is set up to use radioactivity and the throughput is low, then using a radiolabeled assay is still considered the “gold standard” for measuring kinase activity,” says Curtain. If radioactivity is not feasible for you, there are certainly other good options. “The best assays are easy to use and provide data that is biologically relevant,” says Curtain. Some kinase assay examples discussed here, along with challenges in assay design, and some advice from people in the know, can get you off to a good start.
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Kinase assays for high-throughput screening |
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| Molecular Devices offers their kinase assay platform called IMAP for high-throughput screening without the use of antibodies. “Our goal is to enable HTS screening for any kinase in the kinome,” says Boge. Their system is geared toward screening recombinant kinases. “Our IMAP platform is based on the interaction between phosphates and metal-(III)-complex-coated nanoparticles, thus rendering the platform antibody independent. This is a critical advantage with regards to the reproducibility, timing, and speed of screening, in assay development. It a straightforward homogenous mix-and-read assay that uses 5-carboxy fluorescein or 5-TAMRA labeled peptides as substrates.”
Molecular Devices offers more than 150 fluorescently labeled peptide substrates, most of which are validated with their IMAP technology, meaning that they have published the optimal buffer conditions for binding. In response to customers’ difficulties in finding the best substrates for their assays, Molecular Devices developed IMAP Substrate Finders, small groups of peptide substrates sorted by their optimal buffer conditions into 384 well plates. “The different substrate finder plates are dedicated to different parts of the kinome: One for tyrosine kinases, and two different ones for serine/threonine kinases,” says Boge. “The first serine /threonine kinase Substrate Finder covers CAMK and ACG kinase families; the second covers the CMGC/CK1/STE/TKL kinase families. In identifying the substrates for the plate, the first criterion we applied was that the candidate had been used as a peptide in a kinase assay. In addition, we tried to have substrate examples for as many different kinases as possible to maximize diversity and the chance of success in finding substrates for novel kinases. We used these substrate finders ourselves on more than seventy different kinases to create the IMAP assay profiles in our Assay Archive.”
Molecular Devices claims that their modular approach to assay development enables their customers to development assays more quickly. “Our platform is truly generic, alleviating the need for antibodies. It is non-radioactive and homogenous, and easily scalable to 1536 well
plates. It is very flexible in regards to several important factors: ATP concentration, substrate concentration, and choice of substrate. In addition, one can choose among various fluorescent labels and two detection modes. I don't know any other platform that is so tailored towards the
customer's needs,” says Boge.
Another choice for high throughput methods is Promega, which offers several types of assay systems, using different detection methods, for measuring the activity of purified kinases. Their luminescent Kinase-Glo assays “have gained widespread acceptance among high throughput users due to their ‘add and read’ format, low false-positive hit rates and the ability to assay a wide variety of kinase-substrate combinations,” claims Curtain. The assay works by quantifying the amount of ATP left in solution after kinase activity. “While Kinase-Glo is linear out to approximately 10 µM ATP, Kinase-Glo Plus is linear out to 100 µM ATP,” says Curtain. “Additionally, we have custom formulations that can accommodate even higher concentrations of ATP.” These assays can be used for a range of kinase-substrate combinations, and the substrates can be peptides, proteins, lipids, sugars, or alcohols. “The use of substrates that are closer to their native configuration ensures that the in vitro assay more closely mimics what is occurring endogenously,” explains Curtain. “Thus the Kinase-Glo assays help obtain better, more biologically relevant results while being very easy to use.” Other kinase assays offered by Promega include their radiolabeled SAM2 membrane-based assays, and fluorescent assays such as the ProFluor Kinase assays.
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Kinase assays using antibodies for high specificity |
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| Although the absence of antibodies confers flexibility on an assay system, the use of antibodies can confer greater sensitivity, which can be useful when enzyme concentrations are low. Cisbio International offers their universal HTRF kinEASE kit, and specific kinase toolbox reagents, for serine/threonine and tyrosine kinases. “The universal HTRF KinEASE kit uses one proprietary antibody and a choice of 3 proprietary substrates,” says Cathy Drexler, HTRF product manager at Cisbio. “To date, more than 107 serine/threonine kinases can be screened using this single kit.” In addition, the toolbox comprises a panel of antibodies labeled with a Cisbio HTRF tracer such as Europium cryptate, as well as substrate catchers such as streptavidin-XL or anti-tag antibodies labeled with XL. The assay can be used with native or recombinant samples, and is suitable for high-throughput experiments.
Drexler says that their assay is flexible and easy to use: “Assay conditions can easily be adapted to the target kinase. The kit has already been validated on more than 100 kinases and the most appropriate substrate has been determined for each, making assay development very straightforward. On the other hand, the toolbox allows any substrate to be used.”
Also offering a kinase assay with antibodies to provide high selectivity is Invitrogen. Among the many types of kits they offer, covering a range of applications, their PhosphoELISA kits quantify the level of phosphorylation at a specific site on a kinase or kinase target. A similar kit in microarray format can be found in their Mercator PhophoArray kit. In addition, their Omnia kinase assays measure kinase activity with the help of a fluorescent peptide whose signal depends on its phosphorylation state. Mark Surby, a research scientist at Invitrogen, says that “the Omnia platform is one of the only commercially available kinase assays that allows for data collection in real-time, making it ideal for mechanistic evaluations of kinase activity. We are currently extending our Omnia assays into plate- and bead-based immunoprecipitation formats, which will allow our customers to assay the activity of a specific kinase captured from a complex cell lysate. This allows for the capture of entire protein complexes and to measure an activity that is closer to that which exists within the cell.”
Surby believes that the Omnia platform is ideal for studying enzymology: “Our Omnia assays are especially useful for beginners since their real-time nature allows for much faster assay development than standard, endpoint assays. By collecting an entire time course within a single assay well, the researcher can quickly see any adjustments that may have to be made to improve the performance of the assay for their particular application.”
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Challenges in assay design |
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| Kinase assays are continually getting better as their users ask vendors for more options. Molecular Devices found that because each of their clients wanted to perform assays differently, they had more demand for a modular platform on which to run their high-throughput screening assays. “Our challenge in developing these assays, is to enable each company to run the assay according to its specific philosophy,” says Boge. “That is the reason we chose this modular platform approach – to enable our customers to run IMAP according to their specifications. We believe IMAP gives excellent results due to its fidelity, stability and flexibility, in addition to the aforementioned reasons. Ultimately, we believe that in the screening environment ‘convenience’ is very important to the end user because it translates into increased efficiency of the workflow.”
Cathy Drexler, HTRF product manager at Cisbio International, believes that the most challenging aspects of designing or executing kinase assays are the sensitivity of the assay to the inhibitory effects of ATP at certain concentrations, the ease with which assay conditions can be adapted, and the low positive rate. “ATP concentration is a key factor for in vitro assay design because many inhibitor leads for many kinases act by binding the ATP site. Thus, most of the screens are done at ATP concentrations around the Km of the enzyme to identify both potent and moderate inhibitors. To assess the ATP competitiveness, IC50 of the inhibitor is then measured over a range of ATP concentrations. For example, increasing the ATP concentration far above the Km, ATP can be a means to bias the outcome of the high-throughput screening against ATP analogues and to focus the screen on other types of inhibitors such as allosteric modulators.”
Another consideration is the physiological environment of the kinase you are interested in – for example, perhaps it functions in conjunction with protein complexes in vivo. What can you do to adapt the kinase’s in vitro conditions? Drexler submits that this quandary may be important “for understanding the biochemistry and biology of the target, i.e. is the kinase active alone or associated with other proteins? The possibility of using either a peptide, which is generally easy to make and readily accessible to modifications, or physiological proteins, can enable a variety of favorable assay designs for compound inhibitory studies.” Hopefully, consideration of the issues discussed here will help to set you on the right track to choosing a kinase assay and its platform for your own work.
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Caitlin Smith
Contributing Writer
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