| Assay Method Information | |
| | Fluorescence Polarization (FP) Assay |
| Description: | The FP assay uses a tetrapeptide with a sequence arginine-isoleucine-phenylalanine-serine (RIFS) with fluorescein attached to the C-terminus as tracer that was previously shown to bind to the Ubr-boxes of Ubr1 and Ubr2 with similar binding affinity. Assays were developed in 384-well plates, which were read on a Clariostar Reader. Signal-to-background ratio (S/B) was optimized by varying concentrations of GST-Ubr-box and tracer concentrations. GST-fusion proteins were used due to its larger size than Ubr-box alone to increase the FP effect. Purified GST was used to ensure that the tracer does not bind to the GST portion of the fusion protein. The optimized condition includes receptor concentration [GST-UBR1]=2 μM, tracer concentration [tracer]=9 nM, and the highest concentration of unlabeled peptidomimetic compound competitors at 200 μM with 3× serial dilutions. Effective displacement of tracer by unlabeled tetrapeptide confirmed that the fluorescein tag did not significantly alter the binding mode of tracer. |
| Affinity data for this assay | |
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