Miniaturization of Protein Kinase Assays in the MultiScreen®HTS 384-Well P81 Phosphocellulose Filter Plate
Poster Sep 23, 2005
Gertrud Beterams, Steven Sheridan, Chris Barbagallo, and Sonia Gil
AbstractThe protein kinase superfamily contains a significant number of potential targets for pharmaceutical discovery. High throughput screening using filter binding has successfully identified a numberof kinase inhibitors. A new 384-well P81 phosphocellulose (PH) filter plate allows for quantitative protein kinase assays in a high throughput format.
In this study, using Protein Kinase A (PKA) as are presentative assay system, peptide phosphorylation reactions carried out in 384 PH filter plates showed dependence on enzyme and substrate concentrations, and on time. Kinase inhibition studies demonstrated IC50 and Z’ values equivalent to those obtained in 96-well plates. The higher throughput 384 well format offers a miniaturized version of the 96 well assay with equivalent sensitivity and reproducibility.
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