Enzyme-linked immunosorbent assay for the measurement of JNK activity in cell extracts.
A colorimetric enzyme-linked immunosorbent assay (ELISA) for the measurement of kinase activity of c-Jun N-terminal kinases (JNKs) in cell extracts is described. The assay involves passive immobilisation of the substrate GST-cJun on the surface of a microtiter plate, selection of JNK protein kinases directly in substrate-coated wells, kinase reaction, and detection of substrate phosphorylation by a phosphoepitope-specific antibody. The ability of this assay to selectively measure JNK activity relies on the high-affinity interaction between JNKs and c-Jun. Accordingly, we found that JNKs could be captured on the microtiter plate surface through binding to the immobilised GST-cJun. Moreover, JNKs retained the specificity of their interaction with and phosphorylation of c-Jun with respect to the dependence on both intact docking domain and the dimerisation state of c-Jun. This novel procedure represents a marked improvement on conventional radioactive assays in terms of sensitivity, accuracy of evaluation, low time consumption, high throughput and amenability to automation. It is expected to be useful forthe acceleration and facilitation of JNK activity measurement in cell extracts, in particular for large-scale screening of clinical samples.
Tamaskovic
R
R
Institute of Medical Radiobiology of the Paul Scherrer Institute and the University of Zürich, Villigen, Switzerland.
Forrer
P
P
Jaussi
R
R
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002457:Cell Extracts; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005982:Glutathione Transferase / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases; D007700:Kinetics; D051379:Mice; D020928:Mitogen-Activated Protein Kinases; D010766:Phosphorylation; D013379:Substrate Specificity
D002457:Cell Extracts; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases