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Updated: Aug 10, 2026
Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
Assaying protein kinase activity
1Department of Cell and Developmental Biology, Vanderbuilt University School of Medicine, Nashville, TN, USA.
Abstract:
Protein kinases, encoded by approx 2% of eukaryotic genes, represent one of the major classes of cell-regulatory molecules. Assessment of the catalytic activity of a specific protein kinase can be an important step in elucidating signal-transduction pathways that affect cell behavior. As an example of approaches taken to measure protein kinase activity, this chapter presents methods useful for determination of the activity of the oncogenic protein-tyrosine kinase v-Src. Included are protocols for heterologous expression of the kinase in yeast Saccharomyces cerevisiae, immunoaffinity purification from yeast cell lysates, kinase reactions using incorporation of 32P into peptide substrates, and quantifying protein kinase activity. The Notes section discusses alternative approaches for assaying the activity of Src recovered from vertebrate cells and it gives recommendations for assaying the activity of the other protein kinases with respect to the substrate specifity and the composition of kinase reaction buffer.
Insights
This study details methods for measuring protein kinase activity, focusing on the oncogenic protein-tyrosine kinase v-Src. Protocols cover expression, purification, and activity assays, crucial for understanding cell signaling pathways.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Protein kinases regulate crucial cellular functions and are encoded by approximately 2% of eukaryotic genes.
- Assessing protein kinase catalytic activity is vital for elucidating signal-transduction pathways.
- The oncogenic protein-tyrosine kinase v-Src serves as a model for studying kinase activity.
Purpose of the Study:
- To present methods for determining the activity of the protein-tyrosine kinase v-Src.
- To provide protocols for heterologous expression, purification, and kinase assays.
- To offer guidance on assaying other protein kinases, considering substrate specificity and buffer conditions.
Main Methods:
- Heterologous expression of v-Src in yeast (Saccharomyces cerevisiae).
- Immunoaffinity purification of v-Src from yeast cell lysates.
- Kinase reactions involving 32P incorporation into peptide substrates and activity quantification.
Main Results:
- Established protocols for the expression and purification of active v-Src in yeast.
- Demonstrated methods for quantifying protein kinase activity using radiolabeled substrates.
- Provided a framework for adapting these methods to other protein kinases.
Conclusions:
- The presented methods enable robust assessment of protein kinase activity, exemplified by v-Src.
- Accurate measurement of kinase activity is essential for dissecting complex cell signaling networks.
- Recommendations are provided for optimizing kinase assays based on enzyme and substrate characteristics.
