by Caitlin Smith
In vitrokinase assays—in which the biochemical activity of a kinase is measured outside a biological system—have rapidly adapted to modern technology. Traditionally, these assays quantified kinase activity using radiolabeling, which allows high sensitivity and universality (it’s easy to label diverse molecules). Unfortunately, radiolabeling was also dangerous (for the researchers handling the radioisotopes) and expensive (because of waste-disposal cost). Scientists are finding alternatives that are safer and less expensive, but it is still challenging to create alternative assay formats that rival the sensitivity and universality of radiometric-based assays, according to Michael Curtin, product manager for in vitro assays at Promega. Luminescence, fluorescence and antibody-based ELISAs are all expanding the types of in vitrokinase assays available today.
According to Lee Tessler, product manager for discovery and development solutions at EMD Millipore, biochemical kinase activity assays are not an endpoint but rather an entryway into further understanding of complex biological systems. “Follow-up studies must be able to seamlessly draw from cell-based tools, signaling pathway analysis, safety and toxicity profiling and systems biology,” he says. “This requires expertise in these cross-disciplinary methods and the speed and flexibility to execute new assays and interpret multidimensional content.” It’s a tall order indeed, but emerging tools for in vitrokinase assays will speed you toward that goal.
Decide what conditions you need
What equipment is available to you, and what can you purchase? What are your throughput and sensitivity needs? For example, EMD Millipore has two new assay formats with different requirements. Its assays based on homogeneous time-resolved fluorescent (HTRF) resonance energy transfer are designed for PI 3-kinase and tyrosine kinases. “The tyrosine kinase assay employs two proprietary components, the well-characterized 4G10 anti-phosphotyrosine antibody and KinEASE peptide substrates, each of which has been optimized to serve as a substrate for a wide variety of kinases,” says Lucas Armstrong, R&D manager for cellular assays at EMD Millipore. “EMD Millipore also offers in vitrokinase assays that utilize native substrates to better reflect physiological interaction between a kinase and its substrates. EMD Millipore offers Rho-associated Kinase (ROCK) Activity Assay, which employs a native recombinant MYPT1 protein substrate for ROCK.”
HTRF assays are suitable for high-throughput experiments, notes Armstrong, but radiolabeling can yield more sensitive results. “HTRF assays require fluorometers with time-resolved fluorescence capabilities,” he says. “Fluorescence polarization assays are also highly amenable to high-throughput applications but require fluorometers with polarized filters. Radioactivity-based assays require the most handling but are often the method of choice for smaller numbers of samples, if high sensitivity is required.” Another key factor is starting with high-quality, fully-active recombinant kinases, Armstrong says. “The commercial options for in vitrokinase assays as a whole are relatively strong, although customers indicate that there is still room for improvement in assay reproducibility.”
Tessler notes that EMD Millipore’s kinase services provide data with a short (one-week) turnaround time. “Our KinaseProfiler™ service measures kinase functional activity in a fluorescence-free format, enabling quantitative and direct assessment of activity and inhibition,” says Tessler. “Integrated follow-up studies with FlexLabSM custom services provide biological insights through cell-based assays and safety and toxicity profiling. To understand the signaling effects associated with kinase biology, we provide the phosphorylation cascade SignalProfiler™ service. Finally, the DART™ data analysis software package included with our services puts network analysis tools at researchers' fingertips.” EMD Millipore’s FlexLab service scientists can help you coordinate your in vitro kinase assay results with downstream activities such as cell-based assays, cell-signaling measurements and safety and toxicity assessments. Their goal is to help you put your in vitroassay results into a physiologically relevant context.
The InstantOne™ cell signaling assays from eBioscience provide the ease and convenience of a one hour, one wash assay while providing the flexibility to assay both both total and phospho levels in either a 96 well or a 384 well format. According to Matt Slater, senior manager in marketing for assays portfolio at eBioscience, customers have confirmed that their assay is easier than HTRF and western blot assays, while providing more sensitivity than the former, and comparable sensitivity to the latter, respectively. “The TR-FRET assays are more difficult than this assay, even though the TR-FRET assay is no-wash,” says Slater. “I’d rather do one wash than have all these other things to do for TR-FRET-based assays. In addition, with the no-wash assays, you give up some sensitivity, whereas with our assay, you do not give up any.” They recently launched a 384-well InstantOne cell signaling assay in response to customers who used the 96-well plate assays, and said they wanted to do more assays simultaneously. Slater further adds that the InstantOne cell signaling assay is much cheaper than western blots. “People sometimes forget how much westerns actually cost, when you add up all the components to the assay, including parts such as antibodies, gels, membranes, ECL reagents, and film,” he says.
Decide what information you need
Will you measure kinase activity (for which high-quality kits are widely available), or do you need information beyond this? Perhaps you need other measurements—say, the binding portion of the kinase reaction cycle, for which Life Technologiesoffers new tools for study. The company’s LanthaScreen® Eu Kinase Binding Assay is easy to use and well suited to standard IC50 determinations. “However, more importantly, it uniquely enables a number of key applications,” says Steve Riddle, senior staff scientist at Life Technologies. “For example, almost all other platforms measure activity and therefore measure the effect of compounds on the ‘active’ state of a kinase. The LanthaScreen Eu Kinase Binding Assay format measures binding, rather than activity, and can thus be used to evaluate nonactivated kinase states to which a number of important kinase inhibitors bind preferentially (e.g., imatinib). In addition, this format provides a relatively easy means of studying slow binding and slow dissociating compounds.” Life Technologies’ Omnia® Kinase Assays enable researchers to continuously monitor the phosphorylation of peptide substrates, which is useful for enzyme kinetics and mechanistic studies.
Although many vendors offer high-quality kinase assay platforms, Riddle advises choosing a format that is flexible enough to derive additional information about binding kinetics and activation-state preference, in addition to standard affinity measurements. “Such flexibility enables screening for potentially novel chemotypes as well as allows for discrimination of otherwise similar compound sets,” he says.
Decide whether you need additional tools
Do you need a custom combination of kinase assays?
Novus Biologicals’ Mix-and-Match Pathway Assay Kits lets you make your own kit, choosing from eight different analytes, or select one of nine pre-selected kits. “Our kits allow customers to identify both active and inactive forms of many important kinase pathway proteins, such as beta-Catenin, AKT1 and AKT2 and GSK3,” says Stephanie Peacock, director of marketing at Novus Biologicals. “The kits contain all the antibodies and reagents necessary to run the experiment and come in easy-to-use, color-coded ELISA trays.”
Do exercise caution when choosing your assay tools. Peacock notes that not all antibodies are created equal. “Some antibodies that claim to be specific for a particular form (active or inactive) of protein are not actually capable of distinguishing between the different forms,” she says. “Our antibodies have been heavily tested and optimized for use in our kinase pathway kits, and customers can use them to accurately identify various pathway life stages and events. The color-coded, pre-coated wells and vials make it nearly impossible to mix up or confuse your samples. There is no guesswork on which antibodies to pair, what concentrations to use or whether the kit will be sensitive enough to detect the proteins of interest.”
Symansis' Multi-kinase Kinase Array (MKA) assays are unique, phosphospecific ELISA assays that comprise phosphospecific antibodies on eight-well strips for 96-well, strip-plate ELISA layouts. If you want variation but not customization, the company also offers mixed-analyte layouts. “Symansis hopes to have many, many different phosphospecific analytes on strips (up to 150 or more) eventually,” says Lyndon Foster, a scientist at Symansis. “Currently the first, most popular eight analytes are available, including our exclusive AKT1(pS473), AKT2(pS474) and active Beta-Catenin strips, as well as GSKalpha(pS21), GSKbeta(pS9), ERK1/2 (pT202/Y204; pT185/Y187), p38(pT180/Y182) and CREB(pS133).”
The MKA allows you to assay for activation of multiple kinases simultaneously on a single ELISA plate; its strip-plate format lets you mix and match assays for up to six different kinases on a single plate. “Each kit contains a microtiter ELISA plate with 12 removable eight-well strips and all reagents needed to perform the assays,” says Foster. “Each strip of eight wells contains antibodies for a specific kinase. The strips are color-coded, so you can easily identify which kinase assay it is used for.” The MKA has advantages vs. traditional Western blots. According to Foster, MKA kits are easier and faster to use, have greater dynamic range and sensitivity, can run much smaller samples (5 to 20 µg total protein) and are more cost-effective. “In particular, quantitation ELISAs are far superior in the ‘gray areas,’ or in detecting subtle changes in phosphorylation that traditional Western blot development just cannot match,” Foster says.
Do you need downstream resources to put your in vitro kinase assay results into physiological context?
Symansis offers tools for this, too. “When one studies kinase activity, one will eventually need to change or perturb (stimulate or inhibit) the system to ‘see how it works,’ and one will eventually need to examine key biomarker or metabolic endpoint targets like insulin or cytokines that are the ultimate ‘effectors’ of biochemical pathways,” says Foster. “Symansis is unique in that we combine stimulators (exclusive human growth factors and cytokines) and kinase and pathway inhibitors (many of which are exclusive, and 80% are in current clinical trials, for instance) with phospho-ELISA arrays and key biomarker immunoassays to provide researchers with a whole package of reagents designed for studying cell signaling in health and disease.”
Do you need an assay that can be used with a wide range of kinases?
Promega offers the ADP-Glo™ Kinase Assay, which couples kinase activity with a luminescent signal. The ADP that forms during the kinase reaction is converted into light using luciferin and the firefly enzyme luciferase (Ultra-Glo™ Luciferase). “The assay is well suited for measuring the effects that chemical compounds have on the activity of a broad range of purified kinases, making it ideal for both primary screening as well as kinase selectivity profiling,” says Curtin. “The ADP-Glo Kinase Assay can be used to monitor the activity of virtually any ADP-generating enzyme (e.g., kinase or ATPase) using up to 1 mM ATP. We have also introduced 70-plus Kinase Enzyme Systems that are optimized for use with our ADP-Glo platform.”
In vitro kinase assays are an important tool in finding potentially novel therapeutics, but they still require better downstream testing. According to Riddle, “the primary limitation of in vitro kinase assays is their ability to predict in vivoefficacy. More detailed studies of compound mechanisms can help close the gap, but nevertheless there will remain a need for robust and physiologically relevant cellular model systems.”
The image at the top of the page is from Life Technologies' LanthaScreen® Eu Kinase Binding Assay.