NanoLuc-CRBN Fusion Vector
Protein degradation via small molecules such as PROTACs or molecular glues is governed by multiple steps. The first step involves entry of the small molecule degrader compound into the cell, followed by binding to the E3 ubiquitin ligase and protein target. The NanoBRET Target Engagement (TE) Intracellular E3 Ligase Assays allow measuring degrader compound binding or affinity for an E3 ubiquitin ligase protein, Cereblon (CRBN) or von Hippel-Lindau disease tumor suppressor (VHL), in live cells. In addition, the assays can be used to quantify intracellular availability of the compound. These assays are based on the NanoBRET technology, an energy transfer technique designed to measure molecular proximity in living cells.The NanoLuc E3 ligase fusion vectors are designed to be the energy donor in a NanoBRET TE assay that measures the affinity of degrader compounds for a target E3 ligase in cells. The energy acceptor in a NanoBRET TE E3 ligase assay is a fluorescent cell-permeable NanoBRET Tracer that binds to the NanoLuc E3 ligase fusion protein. The close proximity of the tracer to NanoLuc in the E3 ligase fusion results in a NanoBRET signal. Addition of compounds to cells that bind the E3 ligase results in a dose-dependent decrease in NanoBRET signal. This data allows quantitation of the intracellular affinity of degrader compounds for a target E3 ligase protein.The NanoLuc-CRBN Fusion Vector contains the coding region of human E3 ligase component cereblon (CRBN) fused to the C-terminus of the NanoLuc luciferase coding region. The vector can be transfected into various cell lines for CRBN target engagement analysis using the NanoBRET Target Engagement (TE) Intracellular E3 Ligase Assay CRBN. In this assay, it is recommended that the NanoLuc-CRBN Fusion Vector be used with the DDB1 Expression Vector (N2761). The NanoLuc-CRBN Fusion Vector is supplied transfection-ready. The CMV promoter drives the expression of the full-length NanoLuc-CRBN fusion protein. For more information see the NanoBRET TE Intracellular E3 Ligase Assays.The VHL-NanoLuc Fusion Vector contains the coding region of human von Hippel-Lindau disease tumor suppressor E3 ligase (VHL) fused to the N-terminus of the NanoLuc luciferase coding region. The vector can be transfected into various cell lines for VHL target engagement analysis using the NanoBRET Target Engagement (TE) Intracellular E3 Ligase Assay VHL. The VHL-NanoLuc Fusion Vector is supplied transfection-ready. The CMV promoter drives the expression of the full-length VHL-NanoLuc fusion protein. For more information see the NanoBRET TE Intracellular E3 Ligase Assays.The DDB1 Expression Vector contains the coding region of human DNA damaged binding protein 1 (DDB1), a component of a E3 ubiquitin ligase complex. The DDB1 Expression Vector is designed for use with NanoLuc-CRBN Fusion Vector that expresses a NanoLuc-CRBN fusion protein and the NanoBRET TE Intracellular E3 Ligase Assay CRBN. The DDB1 Expression Vector is supplied transfection-ready. The CMV promoter drives the expression of the full-length DDB1 protein. For more information see the NanoBRET TE Intracellular E3 Ligase Assays.
