Cellular Assays
The following list of Gal4-reporter assays (so-called "Mammalian-1-Hybrid" or "Mammalian-2-Hybrid" assays) can be employed for general or selectivity screening purposes. The quality and standardisation parameters for each assay are available upon request.
List of available cellular assays (PDF)
For detailed information or a quotation for your particular need please send a mail to info@phenex-pharma.com
Mammalian-1-Hybrid system
The Mammalian-1-hybrid is derived from the Mammalian-2-hybrid setup. Whereas in the latter, the binding partners are defined in their role as interaction, transactivation and DNA-binding components, the Mammalian-1-Hybrid makes use of the fact that all mammalian cell types express a set of Nuclear Receptors and their associated cofactors. Formally correct, the M1H constructs are Gal-DNA binding domain (Gal4-DBD) fusion constructs with a Nuclear Receptor Ligand binding domain (NR-LBD). The reporter construct is a Gal4 minimal promoter driven luciferase reporter. The difference, however is, that the Gal4-DBD - NR-LBD fusion protein by itself cannot initiate transcription and hence is dependent on the presence of other cofactors which are not defined in this system. Therefore, different mammalian cell lines which are employed for this system can yield different results, depending on the cofactor environment.
Principle of Mammalian-1-hybrid assay
Since all M1H constructs from Phenex use homologous LBDs from different NRs,
the same Gal4-DBD and the same reporter in the same cell line, Phenex has
chosen this assay type for comparative binding analysis, e.g. for selectivity
screening against a nuclear receptor panel.
- Liver X Receptor alpha (LXRa) with control agonist T0901317
- Pregnane X Receptor (PXR) with control agonist SR12813
- Peroxisomeproliferator Activated Receptor gamma (PPARg) with control agonist Rosiglitazone
- Retinoic X Receptor alpha (RXRa) with control agonist 9-cis retinoic acid
Mammalian-2-Hybrid system
To have an alternative for the Y2H system and to verify cofactor (CoF) interactions identified in yeast, Phenex uses a classical Mammalian-2-Hybrid system for a more quantitative analysis. In this setup, the NR is usually expressed as a NFkappaB activation domain fused LBD. The cofactor construct is usually expressed as a Gal4-DNA binding domain fusion. Both are transiently transfected into a mammalian cell line and assayed for NR-CoF interaction by a Gal4 promoter driven reporter construct.
Principle of Mammalian-2-hybrid assay
Application examples for the Mammalian-2-Hybrid system:
- Liver X Receptor alpha (LXRa) plus NCoA3 and Liver X Receptor alpha plus NCoR with control agonist T0901317
- Androgen Receptor (AR) plus NCoA3 with control agonist Dihydrotestosterone
- Constitutive Androstane Receptor (CAR) plus NCoA3 with control agonist CITCO
- Estrogen Related Receptor gamma (ERRg) plus PGC1 with control antagonist 4-Hydroxytamoxifen