1. www.chromotek.com F2H ® Assay: Principle and Examples Fluorescent 2-Hybrid (F2H ® ) Assay: A cell-based assay to monitor reversible protein-protein interactions (PPIs) in mammalian cells in real time

2. www.chromotek.com F2H ® Assay: Principle and Examples www.chromotek.com With F2H ® Assay you can: visualize PPIs inside mammalian cells validate your pull-down results or Y2H hits identify interacting protein domains monitor PPI dynamics upon treatments screen for compounds inhibiting or activating PPIs support biochemical PPI analysis by fluorescence imaging Simply use a conventional fluorescence microscope and any GFP- /RFP-fusion proteins!

3. www.chromotek.com F2H ® Assay: Principle and Examples www.chromotek.com Principle of image-based F2H ® Assay 3 Interaction-dependent co-localization of a GFP-tagged bait protein and an RFP-tagged prey protein at the F2H ® interaction spot Zolghadr et al., (2012) Methods Mol Biol. Zolghadr et al., (2008) Mol Cell Proteomics F2H ® mammalian cell line with a defined F2H ® anchoring spot Interaction: Green spot & Red Spot No interaction: Green spot, NO Red Spot

4. www.chromotek.com F2H ® Assay: Principle and Examples Fluorescent 2-Hybrid (F2H ® ) Assay: Applications

5. www.chromotek.com F2H ® Assay: Principle and Examples www.chromotek.com F2H ® applications: Deletion analysis of PPIs Complete interacting prey protein: Green spot and Red spot co- localize Interacting domain: Green spot and Red spot co-localize Deletion of interacting domain: Green spot is visible; no Red spot Full-length prey Prey domain “p” Prey domain “rey”

6. www.chromotek.com F2H ® Assay: Principle and Examples www.chromotek.com F2H ® applications: Deletion analysis of PPIs 6 Dambacher, S., et al., (2012) Nucleus Mortusewicz, O., et al., (2011) NAR Identification of PPI-domains Meilinger, D., et al., (2009) EMBO Rep Fellinger, K., et al., (2009) J Cell Biochem DNA-ligaseIII-GFPXRCC1-RFP → Interaction → No interaction BRCT domain N-terminal DNA-ligaseIII domains BRCT domain Full-length DNA ligase III N-terminal DNA-ligaseIII domains BRCT domain

7. www.chromotek.com F2H ® Assay: Principle and Examples www.chromotek.com F2H ® applications: Point mutations affecting PPIs Wild-type proteins => Interaction Point mutation in the binding domain disrupting protein interaction => No Interaction

8. www.chromotek.com F2H ® Assay: Principle and Examples www.chromotek.com 8 Identification of disease-related point mutations Detection of interactions from different cellular organelles F2H ® applications: Point mutations affecting PPIs

9. www.chromotek.com F2H ® Assay: Principle and Examples www.chromotek.com F2H ® applications: Profiling of PPI inhibitors 9 Principle

10. www.chromotek.com F2H ® Assay: Principle and Examples www.chromotek.com F2H ® applications: Profiling of PPI inhibitors 10 F2H ® Assay: fully reversible sensitive cell based reliable screening assay Visualization of inhibitor’s kinetics: -p53 and Mdm2 are interacting -addition of Nutlin 3 -disruption of the interaction is imaged in real time: red fluorescence fades from the spot

11. www.chromotek.com F2H ® Assay: Principle and Examples www.chromotek.com F2H ® applications: Profiling of PPI inhibitors 11 Correlation of cellular F2H ® results with biochemical ELISA data Compounds active in F2H ® correlate with ELISA data, although ELISA identified more hits. However, subsequent analyses showed that F2H ® - negative compounds were not cell permeable. Thus the F2H ® Assay directly combines both results. Identification of intracellular active (cell penetrating) compounds. Simultaneous data on cytotoxicity of compounds Dose-response analysis of selected compounds with F2H ® p53/Mdm2 F2H ® screen for inhibitors p53/Mdm2 F2H ® dose-response Yurlova, L., et al., (2014) JBS Brown, C., et al., (2013) ACS Chem Biol

12. www.chromotek.com F2H ® Assay: Principle and Examples F2H ® Assay works with proteins from various compartments

13. www.chromotek.com F2H ® Assay: Principle and Examples www.chromotek.com Use F2H ® to analyze interactions between proteins from different cellular compartments Nuclear proteins: XRCC1 – PCNA PARP1 – PARP2 Nuclear/cytoplasmic proteins: Vimentin – nuclear HZFH β- catenin – TCF4 p53 – Mdm2 p53 – Mdm4 Mitochondrial proteins: DDP1 – TIMM13 In total, F2H® is verified for more than 60 different PPIs (full length, domains, deletions or point mutations) from nucleus, cytoplasm or mitochondria in mammalian cells F2H ® applications: Compartments

14. www.chromotek.com F2H ® Assay: Principle and Examples F2H ® is easy: Experimental procedure

15. www.chromotek.com F2H ® Assay: Principle and Examples You get the F2H ® cell line You get the Platform Reagent You have your GFP-bait and RFP-prey plasmids F2H ® is easy: Experimental procedure

16. www.chromotek.com F2H ® Assay: Principle and Examples F2H ® is easy: Experimental procedure No interaction Interaction

17. www.chromotek.com F2H ® Assay: Principle and Examples www.chromotek.com Advantages of the F2H ® Assay 17  New robust and sensitive HCS assay to measure and quantify PPIs in living cells  Image-based co-localization analysis of fluorescently labeled bait and prey proteins at F2H ® spot (Positional Protein-Protein Interaction Biosensor)  Verified for >60 different PPIs (full length, domains, deletions or point mutations) from different cellular compartments  Very low rate of false positives/ false negatives (< 1%)  Direct and simple optical readout: “Seeing Is Believing!”  Fully functional for reversible interactions  Additional information on e.g. cell permeability or cytotoxicity of compound  Compatible for automated screening assays (Z’ > 0.6)

18. www.chromotek.com F2H ® Assay: Principle and Examples www.chromotek.com 18 contact us: ChromoTek info@chromotek.com +49.89.78 79 73 10