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LanthaScreen Eu Kinase Binding Assay Technology Overview
This easy-to-optimize assay is ideal for screening and multiple downstream applications.
On this web page, you will learn about the kinase binding assay principle as well as different types of applications this assay can enable.
- View kinase compatibility and assay component selection table and kinase-specific protocols.
- Ready to use the assay? Calculate reagent volumes needed for your experiment with our new online calculator.
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Assay overview
With this assay format you can:
- Ensure you’re measuring the right kinase with this intrinsically validated system
- Obtain sensitive detection of kinase inhibitors, including allosterics
- Interrogate active and non-activated kinases
- Track slow-binding inhibitors in real time
- Compare inhibitor on-off rates
Over 200 GST and His-tagged kinases have been tested and validated for use with the LanthaScreen™ Eu Kinase Binding Assay.
See what kinases have been tested and validated with this assay format, view detailed protocols, and learn how to purchase the assay components you need to get started
The principle of the LanthaScreen Eu Kinase Binding Assay is shown in Figure 1. Binding of an Alexa Fluor™ conjugate or “tracer” to a kinase is detected by addition of a Eu-labeled anti-tag antibody. Binding of the tracer and antibody to a kinase results in a high degree of FRET, whereas displacement of the tracer with a kinase inhibitor results in a loss of FRET. Unlike many kinase activity assays, this assay is a simple mix-and-read assay, with no development steps.
Figure 1. Schematic of LanthaScreen Eu Kinase Binding Assay. Any signal resulting from the LanthaScreen Eu Kinase Binding Assay is due to a specific interaction of a Eu-anti-epitope tag antibody bound to the kinase of interest. This ensures no signal will arise due to any other kinases/enzymes present in a test sample, as other kinases or phosphatases present without tag or a different tag produce no signal. In fact, additional kinases or phosphates (with alternative tags or untagged) can be added to a binding reaction to modify the phosphorylation state of the target of interest without need for removal or inactivation.
Invitrogen’s kinase tracers are based on ATP-competitive kinase inhibitors, making them suitable for detection of any compounds that bind to the ATP site or to an allosteric site altering the conformation of the ATP site. Inhibitors that bind the ATP site include both Type I kinase inhibitors, which bind solely to the ATP site, and Type II inhibitors (e.g., Gleevec®/Imatinib, Sorafenib, BIRB-796), which bind to both the ATP site and a hydrophobic site exposed in the DFG-out (non-active conformation). Type III inhibitors are compounds that do not compete with ATP are are loosely referred to as allosteric inhibitors.
A study of 15 diverse Type III inhibitors demonstrated that all but one compound was detected in the binding assay with equivalent potency to activity assays. The sole exception was a substrate-competitive compound, and thus not a true allosteric inhibitor.
Unlike activity assays, the kinase binding assay can be performed with either active or non-activated kinase preparations, making it ideal for detection of Type II inhibitors, as shown in Figure 2. In some cases preferential binding to specific activation states can also be observed for Type I and Type III inhibitors, in addition to Type II inhibitors
Figure 2. Preferential binding of the Type II inhibitor Gleevec® to unphosphorylated Abl.
A. The affinity of Gleevec® was measured using the binding assay by mixing His-tagged Abl (1 nM), both with and without phosphatase treatment, with 0.25 nM of Kinase Tracer 178 and 2 nM Eu-anti-His-Tag antibody followed by TR-FRET measurements. B. The same experiment was conducted using staurosporine in place of Gleevec®.
An additional property of some so-called “allosteric” inhibitors is a slow compound on-rate, due to the compound preferentially binding to a conformation of the kinase that is in equilibrium with other forms. Although this can be measured by monitoring the activity of the kinase at various time points after pre-incubating the kinase and inhibitor, it is substantially simpler to monitor this binding directly, as demonstrated by the binding of BIRB-796 to p38 alpha when binding was directly monitored over time, as shown in Figure 3.
Figure 3. Slow binding of BIRB796. A. Slow binding kinetics was observed for the Type II kinase inhibitor BIRB796. B. The Type I inhibitor SB202190 shows no time dependence.
Most instruments, settings, and filters that work with other europium-based TR-FRET assay systems will perform well with the LanthaScreen Eu Kinase Binding Assay. As with other TR-FRET systems, the europium donor is excited using a 340-nm excitation filter with a 30-nm bandpass. Energy transfer to the Alexa Fluor 647 tracer is measured using a filter centered at 665 nm with a 10 nm bandpass. This signal is then referenced (or “ratioed”) to the emission from europium peak, using a 615 nm, 10-nm bandpass filter. The “emission ratio” is calculated as the 665 nm signal divided by the 615 nm signal.
For assistance with determining if your instrument is suitable to read the LanthaScreen Eu Kinase Binding Assay, or for assistance with instrument setup, please call us at 1-760-603-7200 (select option 3, then enter extension 40266).
* For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
For Research Use Only. Not for use in diagnostic procedures.