SubscribeProtein kinases (PKs) are a diverse group of enzymes involved in many areas of cell signaling. These include cell growth and proliferation and neural functions.
The keen interest in PKs arises from their role in regulating biological mechanisms. Through phosphorylation, PKs participate in many cellular signal transduction processes. Furthermore, defects in these pathways have been implicated in numerous human diseases including cancer, inflammation and diabetes. Research focused on kinase activity could ultimately identify targets that can be used to develop new pharmaceutical agents to treat many of these diseases.
Invitrogen's Far Red PolarScreen™ assays employ a proprietary Far Red fluorophore in homogeneous fluorescence polarization assays. The fluorophore is highly water soluble and, unlike cyanine-based fluorophores, has a fluorescence lifetime that allows for large polarization shifts between free and bound tracer.
Far Red PolarScreen™ kinase assays: the assay method
Fluorescence polarization is a powerful tool with applications in kinase research: FP kinase assays are homogeneous and are amenable to miniaturization, and therefore quite useful in HTS applications. Because FP reports a molecule's tumbling rate, the polarization value relates directly to the molecular volume of the fluorescent molecule. An increase in molecular volume will slow a fluorescent molecule's tumbling rate and yield a high polarization value. Conversely, a decrease in molecular volume will increase the fluorescent molecule's tumbling rate and yield a lower polarization value.
Fig. 1: Schematically illustration of the PolarScreen Far Red assay theory:
Labeled phosphopeptide tracers and any unlabeled phosphopeptide products generated during a kinase reaction compete for binding to antibodies. In a reaction mixture containing no phosphopeptide products, the antibodies will bind a significant portion of the fluorescent tracer, producing a high polarization value. However, in a reaction mixture containing phosphopeptide products, the antibodies will bind a smaller portion of tracer, thereby yielding a low polarization value.
In a Far Red kinase assay (figure 2), kinase, substrate, and ATP are allowed to react in the presence of library compounds. After the reaction is complete, antibody and Far Red labeled tracer are added. The antibody can associate with either the labeled tracer (resulting in a high FP value) or the kinase-produced phosphorylated substrate (resulting in a lower FP value). The amount of antibody that binds to the tracer is inversely related to the amount of phosphorylated product present, and in this manner kinase activity can be detected and measured by a decrease in FP value. Thus, library compounds that inhibit the reaction are identified as wells that have a high polarization value.
Fig. 2: The PolarScreen assay is a simple homogeneous mix-and-measure assay which is highly miniaturizable and much more rapid than ELISA-, heterogeneous fluorometry-based or radioactive kinase assays
High quality fluorescence polarization data
To demonstrate the use of the PHERAstar (figure3) with Invitrogen's Far Red PolarScreen™ kinase assay (#PV3327, Invitrogen, USA), a tyrosine kinase (Csk) titration curve (n = 3) was performed, in the concentration range of 0.5 pg/mL to 2 μg/mL Csk, as shown in figure 4.
Fig. 3: The new high-end multimode microplate reader PHERAstar is designed to perform HTS fluorescence polarization assays in various plate formats including low volume 384-well plates and 1536-well plates
Results were obtained in black low volume 384-well plates (#3676, Corning, USA) with a final assay volume of 20 μL according to the kit protocol.1 During the fluorescence polarization measurement the Far Red fluorophore was excited at 610 nm and the emission was detected at 670 nm in both polarization planes simultaneously.
Fig.4: Tyrosine kinase (Csk) titration with the PolarScreen™ Far Red detection kit on the PHERAstar.
Picogram to microgram quantities of tyrosine kinase (Csk) will phosphorylate sufficient peptide substrate to affect an assay window of between 80 and 280 mP.
Conclusion
Homogeneous fluorescence approaches like fluorescence polarization are important technologies for developing effective HTS assays for discovering therapeutic leads of kinases.
Through this specific PolarScreen ™ Far Red kinase assay example, we have shown that the PHERAstar can produce high quality fluorescence polarization data amenable to screening efforts. The excitation and emission spectra of the far red tracer are red-shifted beyond 600 nm, and thus it shows substantially less interference from light scatter or autofluorescent library compounds than do fluorescein-based tracers.
Seven far-red kinase assays are available to address a wide variety of kinase targets. Far Red kinase assays can be used for both high-throughput screening, as well as further investigation of potential leads to determine accurate EC50 values.
PolarScreen is a trademark of Invitrogen Corporation
References
- PolarScreenTM Tyrosine Kinase Assay Kit, Far Red Protocol #PV3327, Invitrogen, USA
Article by Randy Hoffman, Group Leader, Screening Services, Invitrogen Discovery Sciences
Randy Hoffman is currently the Group Leader of Screening Services at the Invitrogen Discovery Sciences, Madison, Wisconsin, USA headquarters. In this role, Mr. Hoffman oversees the day to day operations of screening customer's compounds in both cell based and biochemical profiling services.