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Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays
Published on: September 19, 2018
Immune-complex assays for tyrosine protein kinases
1Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey, USA.
Current Protocols in Immunology
|April 25, 2008
Summary
This study introduces immune-complex assays to detect tyrosine protein kinases (TPKs) and assess their function. These methods overcome challenges posed by low TPK abundance and rapid activity changes for studying cellular signaling.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Signaling
Background:
- Tyrosine protein kinases (TPKs) are crucial enzymes in cellular signal transduction.
- Their low abundance and rapid activity regulation complicate traditional purification and analysis.
- Immune-complex assays offer a viable alternative for TPK detection and functional assessment.
Purpose of the Study:
- To establish methods for detecting and evaluating the functional state of tyrosine protein kinases.
- To provide protocols that circumvent challenges associated with low enzyme abundance and rapid kinetics.
- To enable the study of TPKs in cellular signaling pathways.
Main Methods:
- Immunoprecipitation of TPKs followed by autophosphorylation and SDS-PAGE analysis.
- Autoradiographic detection of phosphorylated TPKs.
- Alternate protocols utilizing exogenous protein or peptide substrates for functional assays.
Main Results:
- Successful detection and functional assessment of TPKs using immune-complex assays.
- Demonstration of protocols for simultaneous autophosphorylation and exogenous substrate phosphorylation analysis.
- Development of a rapid, high-throughput assay using peptide substrates.
Conclusions:
- Immune-complex protein kinase assays are effective for studying tyrosine protein kinases.
- These methods provide valuable insights into TPK presence, abundance, and functional status.
- The developed protocols facilitate the investigation of TPKs in various biological contexts.
