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In Vitro Kinase and Phosphopeptide Testing (IKPT) Service

Most protein kinases appear to have broad and overlapping substrate specificities and most phosphosites are likely to be targeted by multiple kinases. With so many potential kinases that could phosphorylate critical regulatory phosphosites, it is a major challenge to identify the best candidates. Our In Silico Kinase Match Prediction Services provides a powerful strategy to narrow down the most promising candidate kinases, but this requires further experimental validation for pinning down the very best prospects. Our In Vitro Kinase and Phosphopeptide Testing (IKPT) Services provides an inexpensive means to have a panel of purified and active human protein kinases tested for their ability to phosphorylate a short peptide or a recombinant protein substrate.

Our IKPT Service uses the most reliable direct assay of protein kinase phosphotransferase activity that is known. The methodology is based on the direct quantification of radio-labeled phosphate from ATP (gamma-32P or gamma-33P) on to a peptide or protein substrate of a target protein kinase. This provides for a high signal to noise detection of phosphorylation, high reproducibility. In addition to the measurement of the phosphotransferase activity of a selected kinase against test peptide or protein, we can provide control data with well characterized substrates for the kinase.

The preparations of over 350 recombinant protein kinases that we can use in our IKPT Service possess high specific activities, and generally represent full-length human clones. In some instances, we use kinases that feature activating mutations that may occur in vivo. But generally, the kinases are activated by endogenous phosphorylation in the baculovirus-infected insect cells or by the addition of the purified and activate upstream protein kinase. For each kinase used in the IKPT Service, the assay conditions have been carefully optimized to ensure the highest levels of phosphotransferase activity. The MS-Excel spreadsheet that can be downloaded below features detailed information on most of the protein kinases available with our service along with active hyperlinks to other websites.

Turnaround with our IKPT Service is within 3 weeks of receipt of a peptide or protein for testing. Please add at least two weeks if a custom synthetic peptide substrate is ordered from Kinexus with our Custom Peptide Synthesis Services. Recombinant proteins ordered through our Custom Recombinant Protein Production Services may take another 3 weeks or longer.