ADP Hunter™: A Generic Homogeneous Assay for High Throughput Kinase Inhibitor Screening
Poster Apr 21, 2006
L. Kauffman, V. Shenoy and R.M. Eglen
AbstractThe DiscoveRx ADP Hunter™ assay measures the generation of ADP resulting from kinase phosphorylation of a substrate, in a format specifically designed for the high throughput screening laboratory. ADP is measured through a coupled enzyme system, which is linked to a positive, red-shifted fluorescent readout. The assay has a wide dynamic range (1.2 – 120 µM), and can be used with a wide range of ATP, from less than 5 µM up to 300 µM. No substrate labeling is required, and the assay can accommodate either peptide or whole protein substrates.
The simple assay format, with a single sixty minute incubation, provides robust and reproducible results with signal to background ratios > 10 and typical Z’ values > 0.7. The assay has been validated with several kinases, and against the LOPAC library with excellent results. Compound interference rates are low, and known kinase inhibitor IC50 values are consistent with literature references. In all, the ADP Hunter provides a useful, generic tool for screening novel inhibitors of kinase activity.
We utilized paired synthetic crRNAs coupled with our synthetic tracrRNA in cells transduced with lentiviral Cas9 to perform a functional knockout on hsa-miR-221. This three-part system (crRNA, tracrRNA and Cas9) has demonstrated efficient gene editing when used with only one guide RNA, but the goal was to use two crRNAs to remove the entire stem-loop.READ MORE