TRS Custom Materials
Get started quickly by letting our team do the engineering for you. We specialize in developing traditional reporter vectors and NanoBRET® Target Engagement tracers. Leverage our expertise with a portfolio of innovative bioluminescent solutions designed specifically for your research.
Custom Vectors
Custom vectors can be built to your specifications for a variety of research needs including NanoBRET® Target Engagement (TE), NanoBiT® and NanoBRET® Protein:Protein Interactions (PPI) Assays, and Bioassay development. Vectors can be provided as a stand-alone deliverable for you to do your own assay development or as a milestone step for further assay development. All vectors are compatible with transient expression, with formats for stable cell line generation also available.
Traditional reporter gene assays using:
- Firefly luciferase (luc2, luc2P)
- NanoLuc® luciferase (Nluc, NlucP, secNluc)
- Renilla luciferase (hRluc)
Create protein fusion constructs to express any wild-type or mutant protein of interest (POI) for NanoBiT® or NanoBRET® PPI or NanoBRET® TE assays:
- HaloTag® technology
- NanoLuc® luciferase technology
- NanoBiT® luciferase complementation components: LgBiT, SmBiT, HiBiT
Custom NanoBRET® Target Engagement Tracers
NanoBRET® technology enables the quantitative measurement of target engagement (TE) in live cells with unparalleled precision, allowing you to characterize small molecule engagement within the context of the cellular environment. The NanoBRET® TE method allows you to measure compound affinity and occupancy with the target protein, selectivity in binding to related proteins, as well as compound permeability and compound residence time on the target protein.
The NanoBRET® TE method measures the apparent cellular affinity of test compounds by competitive displacement of a cell-permeable fluorescent NanoBRET® tracer, which reversibly binds to a NanoLuc® luciferase target protein fusion in cells. Promega has developed hundreds of NanoBRET® TE assays for over 340 Kinase targets as well as other key drug targets such as CRBN, RAS and RAF dimers, and components of the DDR pathway.
How We Can Help
If we don’t already have a tracer for your target, our custom team can help! Using our chemistry expertise, we build a custom tracer from your parental compound, utilizing three different linkers to our optimized NanoBRET® 590 Dyes, allowing a panel of tracer candidates to be prepared from a common intermediate and providing the structural diversity needed to ensure the highest probability of success. Assays built with a customer’s proprietary parental compounds are exclusively for the customer and will not be offered to others. We provide you with the total yield of all three tracer variants.
Further develop the NanoBRET® TE assay yourself or work with our custom team on full NanoBRET® TE assay development.
It's important that the choice of the starting compound for tracer synthesis have some SAR or biochemical information which will help in knowing where to add the linker/NanoBRET™ dye without losing all affinity of the compound to the protein. The affinity of this parental compound ideally should be <10nM, if in competition with ATP or <1uM, if no competitor in live cells is expected. Standard delivery is the three tracer variants starting from 20–30mg of an primary amine modified parental compound.
Illustration of the NanoBRET® TE Assay. Panel A: Compound engagement is measured in a competitive format using a cell-permeable fluorescent NanoBRET® tracer. Binding of the test compound results in a loss of NanoBRET® signal between the target protein and the tracer inside intact cells. Panel B: The affinity of the NanoBRET® tracer is determined for each target protein. For analysis of target engagement by a test compound, cells are treated with a fixed concentration of NanoBRET® tracer that is near the EC50 value of the NanoBRET® tracer dose-response curve. Panel C: To determine test compound affinity, cells are titrated with varying concentrations of the test compound in the presence of a fixed concentration (EC50–EC80) of the tracer.
Lumit® Assay Development: Antibody Labeling
We offer a labeling service to complement the Lumit® DIY labeling kit. Yields are generally 80–90% but cannot be guaranteed due to the "stickiness" of the Ab on the Zeba™ clean-up column. Unreacted HaloTag®-BiTs are removed by use of HaloLink™ Resin. Standard QC includes assessment of concentration (as determined after CA addition and purification) and SDS-PAGE characterization. Turnaround time is 2–3 weeks.
Assay Development
- Client provides a description of the desired assay and intended use.
- TRS will schedule a call to get more details, if needed.
- TRS will propose assay options with ballpark pricing and timeline to Client.
- TRS will write a statement of work (SOW) for the preferred option.