Kinase Activity Profiling
Protein kinases are regulatory enzymes that transfer phosphate groups from a donor (usually ATP) to a substrate. This phosphorylation usually leads to a functional change in the target protein (substrate) by altering enzyme activity, cellular localization, or interaction with other proteins. Thus, protein kinases are known to play a key role in cellular processes such as proliferation, apoptosis, angiogenesis or metabolism.
Dysfunctions of protein kinases are the cause of numerous diseases, including cancer.
In order to study kinases in biological systems, the Microarray Core Facility offers a comprehensive service for Kinase Activity Profiling using the peptide substrate microarray platform system PamStation 12 (Pamgene, International). This kinase assay is activity-based and detects protein kinase activity directly in cellular and tissue lysates, through measuring peptide phosphorylation by protein kinases. The resulting kinomic profiles can help elucidate cellular signaling pathways driving particular biological processes and phenotypes and be used to identify and develop biomarkers.
Type of experiment:
- Comparison of cell lines/tissues (e.g. mutant/wt; healthy/diseased)
- Spike in of inhibitor, e.g. in vitro treatment of cell cultures off chip or ex vivo spike-in of cell lysates on chip
- Inhibitor Studies
YOU supply
Important: Sample preparation protocols will be provided on request! Depending on the material from which the lysates are prepared (cell lines or tissue), there are specific protocols that must be applied. Please contact Dr. Melanie Bewerunge-Hudler.
- Completion of sample sheet
- Completion of Study Information Form (Document that provides a summary of the required information for the data analysis).
- Cell Lysates containing active kinases, prepared according to the "Sample Prep" protocol.
- Each condition should be tested in triplicate.
- Provide at least two separate aliquots per sample:
- > 15µl for quality control
- > 25µl for kinase activity profiling (per Chiptyp, e.g. STK-Array)
- (optional) > 25µl for kinase activity profiling (per Chiptyp, e.g. PTK-Array)
Required Protein Concentration:
>200ng/µl (STK-Array)
>500ng/µl (PTK-Array)
- Samples must be provided in a 1.5ml-tube and on dry-ice. Beware: Any freezing/refreezing of the samples should be avoided as this will affect the activity of the kinases!
- It is essential to provide sample sets of 4 samples (or multiples thereof).
We perform
- Assistance with Experimental Design
- Incoming QC for concentration of all samples (Bradford Assay)
- Assay preparation and execution to peptide substrate microarray
- Basic Data Analysis (Software: PamGene’sBioNavigator)
Output to you
A list of peptides (including peptide sequence and uniprot lD) that are differentially phosphorylated, representing the difference in kinase activity between control and conditions. Optionally, upstream kinase analysis.
Please consult the pricelist for detailed cost information.
Selected Publications
Deville, S.S.; et.al.