Build Your Own Assay Tools

Complete Set of Tools to Build Your Own Cell Lines and Cell-Based Assays

Do-It-yourself toolbox products allow you to develop in-house cell lines and cell-based assays for your target and application of interest. Obtain the benefits of using optimized toolbox products for creating your ideal cell line or assay to save you the effort and time required for assay development.

 

Take advantage of Eurofins DiscoverX proprietary Enzyme Fragment Complementation (EFC) technology to develop your own stable cell lines and cell-based assays to study protein-protein interactions, compound-target engagement, cytotoxicity, receptor dimerization and signaling, protein translocation, and much more. Gain an in-depth understanding of the assay principle for consideration of creating optimized assays for multiple applications. Reach out to scientific experts to guide your experiemental design through analysis of your results. Design your workflows and utilize engineered parental cell lines, retroparticles, and plasmid vectors to introduce your target of interest and create cell-based assays for multiple applications.

 

Advantages of Do-It-Yourself Toolbox Products

  • Simple Protocols – Detailed, step-by-step instructions with flow-charts and infographics to easily create assays
  • Flexible Solutions – Utilize any of our cell-line expression vectors to introduce mutants, make modifications, or study unique orthologs or isotypes
  • Any Cell Background – Make your own stable β-arrestin recruitment or cytotoxicity cell-based assays in any dividing cell type with PathHunter® or KILR® retroparticles
  • Complete Kit - Ready-to-use InCELL Pulse™ starter kit includes all the reagents you need to get you started to create your own target engagement assays for your target of interest

Key Resources

Enzyme Fragment Complementation (EFC) Technology Assay Principle

Enzyme Fragment Complementation (EFC) Technology Assay Principle

The EFC technology is based on two recombinant β-galactosidase (β-gal) fragments – a large protein fragment (enzyme acceptor, EA) and a small peptide fragment (enzyme donor, ED), which are added as tags onto proteins in your assay or during the detection step. Separately, the β-gal fragments are inactive, but when combined as part of the assay application (e.g. ligand induced protein-protein interaction), they form an active β-gal enzyme. The enzyme can then hydrolyze a substrate (provided within the detection reagents) to produce a chemiluminescent signal that can be read on any standard luminometer. This simplistic system allows for interrogation of biomolecular interactions for characterizing targets and pathways as well as advancing therapeutic screening and development programs.

Workflow Protocol

When creating your own EFC cell-based assay, you will need the following tools:

  • Cell culture and transfection reagents including tissue culture plates, media, and a facility appointed with proper tissue culture equipment and instrumentation (e.g. a standard luminometer).
  • A plasmid to introduce your target of interest to be tagged with ED. Your target could be a GPCR, kinase, ion channel, transporter, nuclear protein, or other protein of interest
  • Optional: A parental cell line containing a reporter protein tagged with EA. The reporter protein is related to the type of assay being performed. For example, when looking at internalization of a membrane receptor protein to the endosome, the reporter protein would be an endosomal protein. Alternatively, for a GPCR β-arrestin recruitment assay your reporter protein would be β-arrestin.
  • EFC detection reagents that includes the enzyme substrate for producing the chemiluminescent signal.
Easy-to-Follow Protocols for Cell-based Assay Generation Using PathHunter® Parental Cell Lines and Plasmids

Typical workflow protocol schematic to show how to generate an EFC cell-based assay starting with a parental cell line. Start with an engineered PathHunter® parental cell line containing EA to study subcellular translocation (e.g. including membrane receptor trafficking), SH2 recruitment for kinases or cytokine receptors, or GPCR β-arrestin recruitment. The specific parental cell line chosen determines the assay type. Then, create an ED-tagged target protein plasmid followed by transfection of this plasmid into the parental cell line containing EA. Perform antibiotic selection, detect the level of expression of the two fusion proteins (optional), and perform the desired assay in the presence of a ligand.

Toolbox Products

  • PathHunter ProLink™ and ProLabel® Cloning Vectors – Utilize pCMV Mammalian cloning vectors to clone in your protein of interest with a ProLink or ProLabel small β-galactosidase enzyme donor fragment, as an N- or C-terminal tag, and subsequently transfecting into a cell line to create Enzyme Fragment Complementation (EFC) cell-based assays.
  • Expression Vectors – Introduce an expression vector into unique cell types to create EFC cell-based assays. These vectors include a protein target found in our Eurofins DiscoverX cell-based assays tagged with a ProLink or ProLabel sequence.
  • PathHunter Parental Cell Lines – Utilize engineered PathHunter parental cells to rapidly develop β-arrestin recruitment, SH2-protein recruitment, receptor dimerization, or translocation EFC cell-based assays for your protein of interest. These parental cell lines provide the ability to study β-arrestin recruitment with your favorite GPCR, analyze kinase or cytokine receptor dimerization and signaling through SH2-recruitment, or investigate trafficking of your target protein to the plasma membrane, nucleus, or endosome.
  • ChemiSCREEN™ or ChemiBRITE™ Parental Cell Lines – Generate your own stable GPCR cell lines for monitoring calcium or cAMP accumulation. Use with chemiluminescent or fluorescent assays to analyze agonists, antagonists, and modulators of your GPCR target to study functional response. Ready-to-Assay parental control frozen cells are also available.
  • PathHunter β-Arrestin Retroparticles – Make your own stable β-arrestin cell lines and EFC cell-based assays for your target in any dividing cell type. Use the assays to analyze the activation and desensitization of GPCRs and regulation of other signaling molecules such as protein kinases that involve β-arrestin recruitment.
  • KILR Retroparticles – Generate target cells expressing native or engineered surface antigens of choice, in the chosen dividing cell, to measure target cell death in co-cultures. These designer cytotoxicity assays allow you to study antibody dependent cell-mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), antibody dependent cell phagocytosis (ADCP), and other cytotoxicity applications.
  • InCELL Pulse Target Engagement Starter Kit – Easily make your own Compound-Target Engagement cell-based assays to measure inhibitor cellular potency against user-defined protein targets of interest. These assays apply EFC and ligand-dependent thermal stabilization of target proteins such as protein kinases, methyltransferases, and hydrolases. Employ the simple protocol with minimal hands-on time to obtain quantitative inhibitor EC50 values in a native cellular environment.

Create Your Own Trafficking Assays Using Engineered Parental Cell Lines

Create Your Own Trafficking Assays Using Engineered Parental Cell Lines

Generate a PathHunter trafficking assay by first creating a plasmid vector with your target protein of choice tagged with the Enzyme Fragment Complementation (EFC) β-galactosidase (β-gal) enzyme donor (ED). Simply transfect a PathHunter EA parental cell line with the plasmid and perform a trafficking assay in the presence of a ligand. In this example, the Endosome-EA (ENDO-EA) PathHunter EA parental cell line is used, and the target protein is a GPCR. The assay detects the trafficking of the GPCR from the plasma membrane to the endosome upon ligand (pharmacochaperone) binding to GPCR. Once the GPCR moves to the endosome, the ED-tagged GPCR fuses with the large EFC enzyme acceptor (EA) that is bound to an endosomal reporter protein. Upon addition of the β-gal substrate and formation of the active enzyme (ED+EA), the enzyme hydrolyzes its substrate to produce a chemiluminescence signal. For creating a trafficking assay from the endoplasmic reticulum (ER) to the plasma membrane, use the Membrane-EA (MEM-EA) PathHunter EA parental cell line (see an example of this in the following customer publication).

Create Your Own GPCR β-Arrestin Cell-Based Assays in Any Dividing Cell Type

Create Your Own GPCR β-Arrestin Cell-Based Assays in Any Dividing Cell Type

Create your own GPCR β-arrestin cell-based assays to evaluate ligand-induced β-arrestin recruitment to any GPCR in any dividing cell type. Using the Enzyme Fragment Complementation technology, simply infect your target cells with the PathHunter β-Arrestin Retroparticles, transfect the cells with a GPCR plasmid, and perform a PathHunter GPCR β-arrestin assay with your ligand of interest. Note: β-Arrestin2-EA Retroparticles and GPCR-PK can be introduced into the target cells in either order.

Create Your Own Cell-Based Cytotoxcity Assays in Any Dividing Cell Type

Create Your Own Cell-Based Cytotoxcity Assays in Any Dividing Cell Type

Target cells expressing the receptor antigen of choice can be engineered to stably express a protein (housekeeping protein) tagged with the small enzyme donor (called the enhanced ProLabel (ePL) or β-gal reporter fragment) using KILR Retroparticles. When the stable target cell line is used in an EFC-based cytotoxicity assay (e.g., ADCP, ADCC, CDC), the EFC reaction can be measured with the addition of the large enzyme acceptor (EA) β-gal fragment that creates an active β-gal enzyme. With the addition of hydrolyzing substrate, a chemiluminescent output can be detected on any bench top luminometer.