Enzyme-Linked Immunosorbent Assay for the Measurement of JNK Activity in Cell Extracts
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R. Tamaskovic
Abstract
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 for the acceleration and facilitation of JNK activity measurement in cell extracts, in particular for large-scale screening of clinical samples.
Copyright ©1999 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
- Biosynthesis of Glycosylphosphatidylinositols in Mammals and Unicellular Microbes
- Activation of DNA Replication in Yeast by Recruitment of the RNA Polymerase II Transcription Complex
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- Epitope Mapping of the Monoclonal Antibody MM12.10 to External MDR1 P-Glycoprotein Domain by Synthetic Peptide Scanning and Phage Display Technologies
- Molecular Mimicry between a Monoclonal Antibody and One Subunit of Crotoxin, a Heterodimeric Phospholipase A2 Neurotoxin
- Enzyme-Linked Immunosorbent Assay for the Measurement of JNK Activity in Cell Extracts
- Mitochondrial DNA Acts as Potential Promoter of the Baculovirus RNA Polymerase
- In Vitro Phosphorylation of Purified Glycosylphos-phatidylinositol-Specific Phospholipase D
- Cathepsin S and Cruzipain Are Inhibited by Equistatin from Actinia equina
- Lipopeptides as Dimerization Inhibitors of HIV-1 Protease
