There are over 500 protein kinases in the human kinome (Manning et al., 2002. Science 298:1912-34), and thousands of phosphorylation sites in cells (Needham et al., 2019. Science Signaling 12:eaau8645). How do you determine which kinase phosphorylates a particular substrate site of interest? At the MRC PPU, we can now help you answer this question.
A phosphorylation site regulates the activity of your protein of interest. However, the kinase responsible is unknown. In silico methods might provide clues, but they do not account for cellular context and are often unreliable. Genetic screens can be unwieldy and often identify kinases that are only indirectly involved in phosphorylation. You need a direct experimental approach to address this question.
KiPIK screens use chemical kinase inhibitors to identify the kinases responsible for phosphorylation of a substrate in specific cells or tissues. The method uses information from in vitro profiling of hundreds of kinase inhibitors on hundreds of protein kinases. It does not rely on the assumption that one inhibitor inhibits one kinase. This results in superior performance and wider kinome coverage. Screens using the KiPIK approach, developed at Newcastle University (Watson et al., 2020. Nature Communications 11:1684), are now available at the MRC PPU.
We offer a substrate design service and we can generate the required peptide or protein. We can provide custom peptide synthesis and protein production to produce:
Or we can use your own generated peptide / protein as antigen.
We will work with you to optimise phosphorylation of your substrate in cell or tissue extracts of your choice. We will then conduct KiPIK screens in one of two formats:
These assays generate an “inhibition fingerprint” that characterises the unknown kinase activity.
The inhibition fingerprint of your unknown kinase activity will be compared to our database of over 400 kinase inhibition fingerprints. From this analysis we will provide you with ranked lists of candidate kinases for your phosphorylation site, together with statistical indications of significance.
As each investigator’s needs for assay development are different, pricing and turnaround will vary depending on the variables discussed above including:
If you are interested in KiPIK screens, we can help you design your project from the start. We can generate your substrate of interest and even a phospho-specific antibody. Please contact us to discuss your project further and for a quotation!
