Kinase Modification Measurement Service

Radiometric Assays

For ultimate precision and universal applicability, radiometric assays remain the undisputed gold standard for direct kinase activity measurement. These assays utilize radiolabeled ATP, as a phosphate donor. The kinase transfers the radioactive phosphate to a specific peptide or protein substrate, and the resulting phosphorylated product is isolated and quantified via scintillation counting or phosphorimaging.

High-Throughput Non-Radioactive Assays

For large-scale screening campaigns, efficiency and scalability are paramount. We offer a range of non-radioactive, homogeneous "mix-and-read" assays designed for high-throughput screening (HTS).

• Luminescence-Based ATP Depletion: Assays quantify kinase activity by measuring the amount of ATP remaining in a reaction after phosphorylation. The remaining ATP is converted into a luminescent signal by a luciferase enzyme. A lower signal indicates higher kinase activity. This method is highly sensitive and is a workhorse for primary screening.
• Fluorescence-Based ADP Detection: Technologies utilize a highly specific antibody that detects the ADP generated during the kinase reaction. This interaction displaces a fluorescent tracer, causing a detectable change in fluorescence polarization. This provides a direct, real-time measure of enzyme activity.

Mass Spectrometry-Based Phosphoproteomics

To move beyond purified enzymes and understand kinase activity on a global scale within the native cellular environment, mass spectrometry (MS) is the ultimate discovery tool. Our state-of-the-art LC-MS/MS platforms enable deep phosphoproteome profiling.

• Unbiased Discovery: This approach allows for the identification and quantification of thousands of phosphorylation sites simultaneously from cell lysates or tissues, providing an unbiased snapshot of cellular signaling networks.
• Substrate Identification: By combining in vitro kinase assays with MS analysis (e.g., Kinase Assay-Linked with Phosphoproteomics - KALIP), we can definitively identify the direct substrates of a specific kinase, a critical step in elucidating its biological function and the downstream effects of an inhibitor.

Physiologically Relevant Cell-Based Assays

Biochemical assays are essential, but true validation requires assessing a compound's effect within a living cell. Creative Biolabs provides critical cell-based services to bridge this gap.

• Western Blot Analysis: Using phospho-specific antibodies, we can detect changes in the phosphorylation status of a specific target protein within treated cells, confirming on-target engagement in a physiological context.
• High-Content Analysis (HCA) in 2D and 3D Models: We utilize automated microscopy and advanced imaging to quantify phosphorylation events and their downstream consequences (e.g., protein translocation, cell morphology changes) in multi-well plates. Critically, we can extend this analysis to more predictive models like 3D spheroids and organoids, which better mimic the in vivo tumor microenvironment.