Table 17.1 Kinase assay platforms.

Assay Principle References
Adapta® Universal Kinase AssayIn 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 AssaysA 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 AssayBinding 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 AssaysDetection 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 ProteinThis fluorescent ADP/ATP biosensor consists of a recombinant bacterial nucleoside diphosphate kinase site-specifically labeled with an environment-sensitive coumarin dye.7
Omnia® Kinase AssayFluorescence 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 AssayPhosphorylation-dependent protease susceptibility of a double-labeled peptide substrate is detected using FRET.10
CellSensor® Cell LinesCellSensor® 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.
For Research Use Only. Not for use in diagnostic procedures.