Table 17.1 Kinase assay platforms.
| Assay | Principle | References |
|---|---|---|
| Adapta Universal Kinase Assay | In the absence of an inhibitor, ADP formed by a kinase reaction will displace an Alexa Fluor 647 dye–labeled ADP tracer from an Eu3+-labeled anti-ADP antibody, resulting in a decrease in the TR-FRET * signal. In the presence of an inhibitor, the amount of ADP formed by the kinase reaction is reduced, and the resulting intact antibody–tracer interaction produces a high TR-FRET signal. | |
Antibody Beacon Tyrosine Kinase Assay | Peptide substrate phosphorylation is detected via competitive displacement of an Oregon Green 488 dye–labeled ligand from a phosphospecific antibody. | 1 |
| LanthaScreen Kinase Activity Assays | A terbium (Tb3+)– or europium (Eu3+)–labeled phosphospecific antibody binds the phosphorylated fluorescein- or Alexa Fluor 647 dye–labeled peptide substrate, resulting in an increase in the TR-FRET signal. | 2, 3 |
| LanthaScreen Eu Kinase Binding Assay | Binding of an Alexa Fluor 647 tracer to a kinase is detected by addition of a Eu3+-labeled anti–epitope tag antibody. Binding of the tracer and antibody to a kinase results in a high FRET signal, whereas displacement of the tracer by a kinase inhibitor results in a loss of FRET signal. | 4 |
| LanthaScreen Cellular Assays | Detection of phosphorylation or other protein modification event is measured on a TR-FRET–compatible plate reader. Little or no TR-FRET is observed with unstimulated or inhibited cells, whereas stimulated cell samples display high TR-FRET. | 5, 6 |
| NDP Sensor Protein | This fluorescent ADP/ATP biosensor consists of a recombinant bacterial nucleoside diphosphate kinase site-specifically labeled with an environment-sensitive coumarin dye. | 7 |
| Omnia Kinase Assay | Fluorescence enhancement of N- or C-terminal 8-hydroxyquinoline fluorophore (Sox) upon chelation of Mg2+ is coupled to phosphorylation of a peptide substrate at an adjacent Ser, Thr or Tyr residue. | 8, 9 |
| Z′-LYTE Kinase Assay | Phosphorylation-dependent protease susceptibility of a double-labeled peptide substrate is detected using FRET. | 10 |
| CellSensor Cell Lines | CellSensor assays measure pathway-driven activation of transcription factors using GeneBLAzer β-lactamase reporter technology. Minimal amounts of β-lactamase are expressed in untreated cells or cells treated with a pathway-specific inhibitor. Stimulation of the pathway with a ligand or with a constitutively active mutation in a pathway component leads to activation of downstream transcription factor(s), resulting in β-lactamase reporter gene expression. Cells are loaded with a cell-permeable β-lactamase substrate, and β-lactamase reporter activity is measured on a fluorescence plate reader. | 11–13 |
| * TR-FRET = Time-resolved fluorescence resonance energy transfer. 1. Free Radic Biol Med (2009) 47:983; 2. J Biomol Screen (2009) 14:121; 3. Anal Biochem (2006) 356:108; 4. J Biomol Screen (2009) 14:924; 5. J Biomol Screen (2009) 14:121; 6. Anal Biochem (2008) 372:189; 7. Biochemistry (2001) 40:5087; 8. Anal Biochem (2006) 352:198; 9. J Am Chem Soc (2003) 125:14248; 10. Assay Drug Dev Technol (2002) 1:9; 11. Current Chemical Genomics (2009) 3:1; 12. Mol Biosyst (2009) 5:1039; 13. Mol Cancer (2009) 8:117. For further information on these assay technologies and other kinase biology products and services, visit www.invitrogen.com/handbook/kinase. | ||
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