Kinase assay platforms—Table 17.1

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.

For Research Use Only. Not for use in diagnostic procedures.

Table 17.1 Kinase assay platforms.