| Biochemical assays for the characterization of kinases and their inhibitors |
The biochemical assays offered by Kinasera are intended for the characterization of ATP and protein substrate competitive inhibitors of protein kinases. The principle of the assays is competitive displacement of ARC-Photo probe from its complex with a protein kinase by an active site targeted inhibitor. The biochemical assays are also suitable for the
determination of the concentration of active protein kinases by titration of the active sites of the kinase.
Kinasera biochemical assays are suitable for HTS as they are amenable to automation (384-well format)reliable - Z factor > 0.80 (as determined with a PheraStar (BMG) platereader)simple one step mix & read setupquick - incubation time 15 mincheap - no lantahnides, no antibodies, no substrates used
lead compound selection in one point displacement assaydetermination of IC50 and Kd values of kinase inhibitorskinase inhibitor selectivity profilingdetermination of the concentration of the active form of the protein kinase
Kinasera biochemical assays are available in two formats:
Fluorescence Anisotropy-based Kinase-Binding assay (Kinasera-FA assay)
The determination of the amount of ARC-Fluo probe in complex with protein kinase is based on the difference in the anisotropy/polarization values of free and bound ARC-Fluo probe. ARC-Fluo probes with different fluorescent labels (FITC, TAMRA etc.) are available to enable best compatibility with the available fluorescence anisotropy detection system.
Time-resolved fluorescence-based Kinase-Binding assay (Kinasera-TRF assay)
Kinasera-TRF assay uses the ARC-Lum probe which only upon binding to a protein kinase emits long lifetime luminescence. Kinasera TRF assay is applicable with wide range of TRF based detection systems including europium- and terbium-based TR-FRET, HTRF and LanthaScreenTM assays (excitation at 340 nm, emission at 665 nm or 520 nm respectively).
| Kinasera-TRF assay || Kinasera-FA assay |
| Assay setup || Homogeneous, 1-step mix & read; 384-well format || Homogeneous, 1-step mix & read; 384-well format |
| Assay volume || 10 - 25 microL || 10 - 25 microL |
| Detection principle|| Time-resolved fluorescence (TRF)(excitation 337nm, emission in-between 520 and 700 nm)|| Fluorescence anisotropy (FA)(fluorescent dye dependent excitation and emission filters)|
| Ligand || ARC-Lum probe || ARC-Fluo probe |
| Suitable for || determination of affinities of ATP- and protein-competitive ligands of basophilic protein kinases || determination of affinities of ATP- and protein-competitive ligands of basophilic protein kinases |
| Targets || Around 50 protein kinases || Around 20 protein kinases |
| Currently tested for || PKA, AKT3, ROCK I, ROCK II, PKG I, PKC, MSK1, Pim1 || PKA, AKT3, ROCK I, ROCK II, PKG I, PKC, MSK1, Pim1 || Main benefits || TRF-based detection enables the characterization of fluorescent inhibitorsGreat excess of ARC-Lum probe can be used to shift the assay away from the tight binding region of the inhibitorsLess sensitive to nonspecific binding of highly abundant proteins Suitable for measuring off rates of competitive inhibitors|| RatiometricLess demanding apparatuses|