KinaseSeeker is a binding assay based on a patented cell-free split-luciferase technology. Split luciferase fragments fused to a scaffold protein and kinase are translated in vitro in a cell-free system and reassembled into a functional luciferase in the presence of a kinase active site binding probe. KinaseSeeker assay has the following benefits over other assay techniques:
Luminescence detection, hence low false positives from fluorescent compounds
Binding assay, hence identification of active site directed and allosteric inhibitors is possible
Cell-free format, hence ability to identify inhibitors in a complex cellular environment at cellular ATP concentrations
Homogeneous assay, hence not affected by artifacts due to immobilization
Sensitive with Low Background; Broad dynamic range
We currently have 215 protein kinases spanning all families on our panel. We are continuously working to add more kinases. If you would like to stay informed when new kinases are added, please send us an email at email@example.com to be added to our mailing list.
We offer an a la carte service, where you can profile 50 kinases of your choice. In addition, we offer several preset panels, comprising popular kinases, which can be used to profile your compounds cost effectively.
We test all compounds for luciferase inhibition prior to testing for kinase inhibition. If your compound inhibits luciferase in our control assays, we will notify you ahead of initiating profiling experiments. These control experiments are part of the profiling services we provide for kinases. In the event that your compound cannot be tested for kinase inhibition, you will not be charged for the control experiments.
The KinaseSeeker assay entails incubation of the translated kinase with the inhibitor for 1 hour at room temperature in rabbit reticulocyte lysate. We have tested the concentration of ATP in several batches of lysate using Promega’s ENLITEN assay. The ATP concentration was approx. 1 mM, which is well above its Km for most protein kinases.
Upon completion of testing (~10 business days), an electronic copy of the study report is submitted to the customer via email. The screening data is reported as % activity remaining for each kinase and compound profiled, where a lower number indicates a more potent compound.
The turnaround time for a report is ~10 business days from the receipt of sample. We make every effort to complete and send data to you within this time frame, if however for some reason there is a delay, we will inform you ahead of time.
The amount of material needed depends on the experiments that you would like us to run with your compound. In general, we need 200 uL of a 10 mM solution of your compound in DMSO for a profiling run (or a pre-weighed equivalent amount of the solid). Please contact us if you do not have enough material to send us. We can return unused material upon request, if you are willing to pay for the shipping costs.
The cell-free split-luciferase technology is very versatile and has been applied to several biological systems. Among these are protein-protein interactions, DNA damage markers (DNA oxidation, DNA UV-modifications, poly (ADP-ribosylation)), Epigenetics (DNA methylation), and enzymes like Proteases, Kinases and Poly (ADP ribose) glycohydrolase (PARG). Please refer to our list of publications for details.
We welcome inquiries about new targets and can develop custom assays for the target of your choice. If you are interested in collaborating with us, please contact us at firstname.lastname@example.org.
Yes, we can screen compounds against protein-protein interactions of the Bcl-family, p53/MDM2 and Hif1a/p300. For example, we can profile compounds against a broad Bcl-panel, whereby we detect inhibition of binding of the helices of Bim/Bad/Bak/Bik/Bid/Bmf/Puma/HRK group to Bcl2/Bcl-xL/Bcl-w/Bfl/Mcl1. Such comprehensive screens can be useful for garnering selectivity information for Bcl-inhibitors.
Yes, our assays are capable of detecting DNA modifications resulting from oxidative stress (oxo-guanine), UV-exposure (UV-photo adducts) or other forms of damage (which result in poly (ADP-ribose) formation. Our assays are also capable of detecting epigenetic markers like DNA methylation.
The cell-free split luciferase technology is very versatile and can be used for many different applications. If your specific target is not listed above, please contact us to discuss the feasibility of using the assay technology for your application