Custom Assay Development Services

Don’t see the assay you need in our collection? Our TRS team can help! We work with you to understand the goals of your research and target proteins of interest and develop an optimized assay to meet your needs.

1. Using our chemistry expertise, we build a custom NanoBRET® Tracer from your parental compound, utilizing three different linkers to ensure optimal performance. Assays built with a customer’s proprietary parental compounds are exclusively for the customer and will not be offered to others.
2. We design and synthesize a custom vector, inserting your target gene(s) into a NanoLuc® fusion expression vector. Alternatively, we utilize a NanoBiT-based approach to enable the study of compound binding to protein complexes.
3. We determine optimized NanoBRET® assay conditions by evaluating tracer affinity, permeability and assay window. Assays are developed to be quantitative for intracellular compound affinity (or apparent K_i) by determining the NanoBRET® Tracer concentration, which is less than or equal to Tracer K_d.
4. We can optionally test known compounds to demonstrate the expected pharmacology, ensuring your assays are reliable and reproducible.

What You Receive

1. A milestone report describing optimized assay conditions
2. The remaining volume of each tracer
3. Each NanoLuc® protein fusion vector (20µg)

Our assay protocols typically involve transient transfections in HEK293 cells, but we can also create and provide thaw-and-use (T&U) cells from the transiently transfected cell pool. T&U cells allow you to treat cells as reagents, eliminating the need to grow and transfect cells and reducing variability. If you prefer a stable cell line, we can create that for you as well.

Don’t have a parental compound? We can screen your custom vector(s) against our existing panel of tracers to identify potential binding interactions.

Our custom PPI assay development process includes the following steps to build your PPI assay with the best performance characteristics.

1. Vector Synthesis: DNA vectors are designed to create fusions of the target proteins of interest with all possible tag orientations. For cytosolic proteins, this involves creating 8 fusion constructs.

• NanoBiT® PPI Assays: 4 SmBiT + 4 LgBiT protein fusions.
• NanoBRET® PPI Assays: 4 HaloTag® + 4 NanoLuc® protein fusions

Mutant controls can also be included when available. Fusions are created in bidirectional-ready vector backbones to allow for combination into a single construct.

2. Assay Development: We optimize the assay conditions by testing different tag orientations and transfection levels to obtain the combination that provides the most optimal assay window. For NanoBRET assays the optimal ratio of donor:acceptor is also determined.

3. Optional Milestones

• Compound Testing: We can test your compounds against the optimized assay to generate dose-response curves.
• Thaw-and-Use Cells: Our assay protocols typically involve transient transfections in HEK293 cells, but we can also create and provide thaw-and-use (T&U) cells from the transiently transfected cell pool.
• Stable Cell Line Development:  If you prefer, we can create stable cell lines.

What You Receive

• A milestone report describing optimized assay conditions
• Each protein fusion vector (100µg)

Reporter Knock-in Cell Lines

The combination of CRISPR/Cas9 gene editing and bioluminescent protein tagging enables the development of assays for quantitative analysis of protein degrader function on endogenously expressed proteins. 

• Use HiBiT knock-ins to perform quantitative end-point target protein analysis, or design the cell line to be compatible with live cell kinetics to generate key degrader metrics such as degradation rate, D_max, DC₅₀ and protein recovery.
• Use HaloTag®-HiBiT tandem knock-in to understand the suitability and phenotype of target protein degradation using the HaloPROTAC3 degrader.
• Use HiBiT or NanoLuc® knock-in cell lines as a starting point to further characterize ternary complex formation and target ubiquitination levels.

How We Can Help

With our technology expertise and experience in building over 250 knock-in cell lines in 50+ cell backgrounds, our custom team can build you the cell line needed to enable your degrader discovery and characterization.

Targeted protein degraders, such as PROTACs, require binding to both the target protein of interest and the intended E3 ligase to drive degradation. In addition, compound effectiveness is dependent on compound permeability, which can be challenging for the hetero-bifunctional PROTAC compounds that are large and “beyond rule of 5”, when compared to classical small-molecule inhibitors.

NanoBRET® TE Technology provides real-time insights into how targeted protein degraders interact with their target proteins or intended E3 ligase in live cells. Using a bioluminescence resonance energy transfer (BRET) technique, these assays measure the binding affinity of degraders to either the target protein or E3. In addition, the assays enable the assessment of the apparent cellular affinity in live cells and the intrinsic binding affinity in permeabilized cells, where the plasma membrane barrier is removed. This dual-mode approach allows for a comprehensive evaluation of a degrader’s intracellular availability—an essential factor for optimizing the design and efficacy of these compounds.

How We Can Help

Work with our custom assay team to design a NanoBRET TE assay for either your target protein or E3 ligase.

Inducing a protein-protein interaction is fundamental to the function of PROTACs and other degrader compounds. They facilitate the recruitment of an E3 ubiquitin ligase to the target protein to generate a ternary complex, resulting in target protein ubiquitination and subsequent degradation by the proteasome when the ternary complex is productive. NanoBRET® PPI Technology allows for the measurement of ternary complex and ubiquitination within a live-cell environment, allowing researchers to:

• Optimize the design of degraders to enhance their potency, selectivity, and overall efficacy in degrading the intended target proteins
• Fine-tune the molecular structure of degraders to improve their therapeutic potential
• Confirm proposed mechanisms of action

How We Can Help

Work with our custom assay development team to develop assays for either ternary complex or target protein ubiquitination using NanoBRET® PPI technology.

• Develop the assay starting with endogenous HiBiT-tagged proteins. We start with a CRISPR engineered cell line with HiBiT fused to the target protein of interest. Transient transfection conditions are determined for the target E3 ligase/ubiquitin.
• Develop the assay in transient format. We work in HEK293 cells and develop the optimal vectors and transfection conditions for your target protein and E3 ligase/ubiquitin. We primarily develop assays based on VHL and CRBN E3 ligases but can also work with novel E3s upon request.

Often the first step in degrader compound development is understanding how loss of the target protein will impact the cellular response. As an alternative to protein knock-out, HaloPROTAC3 is a small molecule degrader that can be used to drive targeted degradation of proteins expressed as HaloTag® fusions to investigate the cellular phenotype of protein loss. When proteins are tandem tagged with HaloTag-HiBiT, target protein levels can be easily quantified following HaloPROTAC3 treatment, allowing for correlation between target protein levels and observed cellular response.

How We Can Help

With our expertise in HaloTag® and HiBiT technologies and experience in building over 250 knock-in cell lines, our custom team can build you the HaloTag-HiBiT knock-in cell line needed to enable the characterization of target protein loss and help you further understand the biology of the protein being studied.