(See Notice 4) 2.2 Transfer of kinase reaction products to filter paper Aluminum foil. Whatman Grade P81 ion exchange chromatography paper. Pencil and ruler for marking P81 paper. 0.5% phosphoric acid. Improving potency is also important for increasing the likelihood of selective activity against the target of interest. Selectivity is a major challenge in the development of kinase inhibitors since most target the highly conserved ATP binding pocket. Due the conservation of this pocket among all kinases, compounds that bind to this region will often inhibit other kinases as well. Inhibitors that have low potency must be administered at high concentrations to be effectiveCat these high concentrations, off-target effects are more likely to occur, potentially resulting in dose-limiting toxicities. One approach to circumvent these difficulties is to screen large libraries of compounds against large libraries of kinases in an unbiased, target-blind manner (2). The information extracted from such large screens can identify molecular scaffolds that potently inhibit previously unidentified targets, suggest avenues for the repurposing of existing small molecule compounds, and provide critical information about inhibitor selectivity. Such screening efforts necessitate strong and high-throughput methods for carrying out kinase assays. These high-throughput kinase assays are often classified as either binding assays or functional assays. Binding assays measure the physical conversation between a compound and a S18-000003 kinase. Functional assays directly measure the switch in kinase catalytic activity in the presence of an inhibitor. These methods may not yield identical results because, for example, compounds can bind a kinase but not inhibit its catalytic activity. We recently reviewed numerous kinase assays that fall into these two classes (1) and both types of assays have been used in recent large-scale S18-000003 screens of small molecule libraries against selections of recombinant kinases (3C6). In this protocol, we describe a high-throughput functional assay, generally referred to as a dot blot kinase assay, that can be readily carried out in a biochemistry laboratory that is equipped and approved for the use of radioisotopes. This radiometric assay entails performing a kinase reaction in the presence of ATP made up of a radiolabeled terminal (gamma) phosphate and a peptide substrate (Physique 1A). The products of the reaction are spotted on phosphocellulose (P81) paper to which the peptide substrate binds. The radiolabeled phosphate group that has been transferred to the bound peptide can be visualized and quantified using a phosphorimager (Physique 1B). In addition to being used for screening, this technique can also be used to carry out dose-response measurements to determine the potency of S18-000003 a particular compound against a kinase of interest. Open in a separate ID1 window Physique S18-000003 1 (A) Radiometric kinase assay. In addition to standard (unlabeled) ATP, ATP made up of a radiolabeled terminal phosphate is included in the reaction mixture. Once the kinase reaction is completed, the radiolabeled phosphate is usually transferred to the peptide. (B) Dot blot capture and visualization of radiolabeled kinase reaction products. Kinase reaction products are spotted on filter paper and washed, removing excess radiolabeled ATP and facilitating peptide binding. A phosphorimager is used to visualize S18-000003 and quantify the counts on the filter paper for each kinase reaction. The dot blot kinase assay is considered a gold standard for assaying kinase functional activity as it directly steps catalytic activity using standard ATP as the phosphate donor. Thus, unlike many binding assays, the dot blot kinase assay closely approximates the reaction. A key advantage of the dot blot kinase assay is the efficiency with which many kinase assays can be performed in parallel; hundreds.