The Fluorescent Two-Hybrid (F2H®) Kits for p53-Mdm2 and for p53-Mdm4 respectively enable intracellular analysis of the interaction between the tumor suppressor p53 and its negative regulators Mdm2 or Mdm4.
GFP-tagged human p53 is anchored at a specific location in the nucleus of F2H®-Cells, forming a bright fluorescent spot in the green channel.
Interaction with the RFP-tagged human Mdm2 or the RFP-tagged human Mdm4 can be easily evaluated by conventional fluorescence microscopy as enrichment of red fluorescence at the location of the green spot.
Compounds’ ability to disrupt the p53 and Mdm2 interaction or the p53 and Mdm4 interaction is determined based on the disappearance of the red spots from the anchored green spots.
F2H® allows direct side-by-side comparison of the compound's activity and kinetics in live cells.
p53:Mdm2 + Nutlin
p53:Mdm2 + sMTide02
p53:Mdm4 + Nutlin
p53:Mdm4 + sMTide02
Incubations with 50 μm Nutlin-3 or stapled peptide sMTide-02 were carried out for 7 h during imaging. Fading of the red fluorescence from the spot but not from the rest of the nucleus indicates disruption of the interaction.