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Updated: Jan 5, 2026
Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays
Published on: September 19, 2018
Assays for tyrosine phosphorylation in human cells
Monica Kruk1, Naomi Widstrom1, Sampreeti Jena1
1Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, United States.
Abstract:
Tyrosine kinases are important for many cellular processes and disruption of their regulation is a factor in diseases like cancer, therefore they are a major target of anticancer drugs. There are many ways to measure tyrosine kinase activity in cells by monitoring endogenous substrate phosphorylation, or by using peptide substrates and incubating them with cell lysates containing active kinases. However, most of these strategies rely on antibodies and/or are limited in how accurately they model the intracellular environment. In cases in which activity needs to be measured in cells, but endogenous substrates are not known and/or suitable phosphospecific antibodies are not available, cell-deliverable peptide substrates can be an alternative and can provide information on activation and inhibition of kinases in intact, live cells. In this chapter, we review this methodology and provide a protocol for measuring Abl kinase activity in human cells using enzyme-linked immunosorbent assay (ELISA) with a generic antiphosphotyrosine antibody for detection.
Insights
Measuring tyrosine kinase activity in live cells is crucial for cancer research. This study reviews cell-deliverable peptide substrates as an alternative method for monitoring kinase activity, particularly Abl kinase, using ELISA.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Tyrosine kinases regulate vital cellular processes.
- Dysregulated tyrosine kinase activity is implicated in diseases, notably cancer.
- Existing methods for measuring kinase activity often rely on antibodies or do not accurately mimic the intracellular environment.
Purpose of the Study:
- To review methodologies for measuring tyrosine kinase activity in live cells.
- To present a protocol for assessing Abl kinase activity in human cells.
- To highlight the utility of cell-deliverable peptide substrates when endogenous substrates or specific antibodies are unavailable.
Main Methods:
- Review of existing tyrosine kinase activity measurement techniques.
- Development and application of a protocol using cell-deliverable peptide substrates.
- Utilizing enzyme-linked immunosorbent assay (ELISA) for detection.
- Employing a generic antiphosphotyrosine antibody for quantifying Abl kinase activity in intact cells.
Main Results:
- Cell-deliverable peptide substrates offer a viable alternative for measuring kinase activity in intact cells.
- The described ELISA protocol enables the assessment of Abl kinase activity in human cells.
- This method provides insights into kinase activation and inhibition within a relevant cellular context.
Conclusions:
- Cell-deliverable peptide substrates are valuable tools for studying tyrosine kinase activity in live cells.
- The presented methodology, using ELISA and a generic antiphosphotyrosine antibody, is effective for measuring Abl kinase activity.
- This approach overcomes limitations of antibody-dependent methods and is applicable when endogenous substrates are unknown.
