The Discoverx PathHunter assay platform provides a screening compatible assay format that is able to directly measure nuclear translocation events without imaging or a reporter gene. This is accomplished using positional complementation in cells engineered to express an inactive β-Gal acceptor fragment (EA) in the cell nucleus and a protein of interest in the cytoplasm that has been tagged with a short complementing peptide for β-Gal called ProLabel. This approach has been applied successfully to a number of targets, but is particularly effective for Nuclear Receptors (NHRs). Using clonal HEK and CHO cell backgrounds with the EA expressed exclusively in the nucleus, we have been able to transiently express NHR-ProLabel fusion proteins and measure translocation events after as little as one hour of compound treatment, often with signal-to-background rations in excess of 10-fold. Data will be highlighted for a series of target, including GR, AR, PXR, LXR and VDR.
Life Sciences 2007 (2007) Proc Life Sciences, PC503
Poster Communications: Application of a homogenous, non-imaging cell-based assay for rapid and direct detection of NHR translocation
A. Fowler1, M. Mathrubutham2, K. Peng2, M. Nguyen2, H. Fabionar2, S. Kuchibhatia2, K. Olson2, P. Grant1
1. Discoverx, Birmingham, United Kingdom. 2. Discoverx, Fremont, CA, USA.
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Where applicable, experiments conform with Society ethical requirements.