The KinaseFinder Screening Service
The KinaseFinder is a biochemical assay intended to identify kinases, which accept a given substrate for phosphorylation.
The KinaseFinder is a standardized service package for identifying protein kinases that can phosphorylate a peptide or protein of interest provided by our customer. Kinase hits, identified with the standard package, should subsequently be confirmed in a second experiment. The KinaseFinder is ideal for the elucidation of physiological pathways with the involvement of protein kinases.
- Potential substrates can be peptides or proteins
- Compare peptides with phospho-site mutations
- Option to follow up with an SDS-PAGE and autoradiogram to visualize the phosphorylated substrate
KinaseFinder Assay Principle
The target substrate is incubated with different kinases in every well of a multi-well plate and 33P-ATP that serves as a phosphate donor.
The kinase catalyzes the transfer of 33P when the protein or peptide is recognized as a substrate.
Substrates are either bound to scintillant-coated ScintiPlate (33PanQinase™) or dropped on filter paper for readout (HotSpot™).
List of Kinase Panels
| Type of KinaseFinder | # of kinases | Assay format |
|---|---|---|
| Tyr protein kinases | 94 | ³³PanQinase™ |
| Ser/Thr protein kinases | 245 | ³³PanQinase™ |
| Ser/Thr & Tyr protein kinases | 339 | ³³PanQinase™ |
| Wild Type Kinase Panel | 380 | HotSpot™ |
| Custom panel | selected by customer | HotSpot™ |
Assay Details
Example data with 50 Ser/Thr kinases.
Activity of kinases with example substrate protein are shown (corrected for substrate background activity). Substrate protein was tested at one concentration. Yellow: Kinase with substrate protein; blue: Kinase without substrate protein (autophosphorylation). A ratio value between substrate phosphorylation and apparent autophosphorylation > 3 may be considered as significant. Performed with the 33PanQinase™ assay. cpm = counts per minute
Setups: Proteins and peptides are accepted as substrates.
Assay format: radiometric screening with 33P-ATP as phosphate donor
Turnaround time: 10 business days for standard projects. Expedited scheduling and data delivery can be arranged prior to commencement of the studies.
Screening location: Assays run with the 33PanQinase™ assay are performed at the German site. Assays run with the HotSpot™ assay are performed at the US site.
Corvin Walter et al. employed the Kinase Finder service to identify kinases that phosphorylate the TOM70, which represents the main import receptor for the carrier import pathway. Kinases DYRK1A and DYRK1B scored highest in phosphorylating TOM70 in the primary Kinase Finder screen (Fig 1a). The hits were confirmed by using increasing concentrations of substrate (Supp Fig. 1b). To confirm the DYRK1 specific phosphorylation site, a TOM70 mutant was used for screening which resulted in significantly less phosphorylation (Supp. Fig. 1g).
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