BIOL 256 |
| Efforts to aid lead optimization using traditional assay formats such as LANCE and DELFIA is often constrained by the limitation of the detection reagents present in the assays. Specifically, the efforts to assess the substrate or ATP-competitiveness of a kinase inhibitor are often times limited by these indirect assays. We used a Nanostream LD µPLC System to develop a separation-based assay to determine the phosphorylation of the substrate by a serine-threonine kinase. This assay allowed direct detection of the phosphorylation product in real time and is devoid of the reagent constraints experienced in other assays. The substrate was labeled with fluorescein at the N-terminus to increase detection sensitivity. The separation of substrate and product was achieved well using reversed phase media and under slight basic pH conditions (8.2). The use of the 24-channel cartridge allowed fast assay optimization and increased throughput of inhibitor dose response titrations. The IC50 determination using 16 well data points translates into a throughput of 1.5 compounds per run of 12 minutes. The rank order of inhibitor potencies, wortmannin and staurosporine, determined by Nanostream LD µPLC System are comparable to those obtained from LANCE. |
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Frontiers in Chemical Biology
5:00 PM-7:00 PM, Wednesday, August 22, 2007 BCEC -- Exhibit Hall - B2, Poster
Division of Biological Chemistry |