scholarly journals Cell surface detection of membrane protein interaction with homogeneous time-resolved fluorescence resonance energy transfer technology

2004 ◽  
Vol 329 (2) ◽  
pp. 253-262 ◽  
Author(s):  
Damien Maurel ◽  
Julie Kniazeff ◽  
Gérard Mathis ◽  
Eric Trinquet ◽  
Jean-Philippe Pin ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Membrane Protein ◽  
Cell Surface ◽  
Fluorescence Resonance Energy Transfer ◽  
Protein Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Membrane Protein Interaction

Development of a Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay for the Inhibition of Keap1–Nrf2 Protein–Protein Interaction

2020 ◽  
Vol 26 (1) ◽  
pp. 100-112
Author(s):  
Sumi Lee ◽  
Dhulfiqar Ali Abed ◽  
Lesa J. Beamer ◽  
Longqin Hu
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Small Molecule ◽  
Protein Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Protein Protein Interaction ◽  
Nrf2 Protein

The transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), plays a major role in regulating the antioxidant defense system through the Kelch-like ECH-associated protein 1–Nrf2–antioxidant response element (Keap1–Nrf2–ARE) pathway. Small-molecule inhibitors targeting Keap1–Nrf2 protein–protein interaction (PPI) decrease the rate of Nrf2 degradation by the 26S proteasome and thus increase the intracellular level of Nrf2, which translocates into the nucleus, leading to upregulated expression of cytoprotective and antioxidant enzymes. Such inhibitors can be developed into potential preventive and therapeutic agents of diseases caused by oxidative damage. To more effectively identify promising Nrf2 activators through the inhibition of Keap1–Nrf2 PPI, a homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assay was developed in this work by indirectly labeling the Keap1 Kelch domain protein with Tb-anti-His antibody as the donor and using, as the acceptor, fluorescein isothiocyanate (FITC)-labeled 9mer Nrf2 peptide amide, the same fluorescent probe that was used in an earlier fluorescence polarization (FP) assay. Assay conditions, including concentrations of the various components, buffer type, and incubation time, were optimized in the TR-FRET competition assay with known small-molecule inhibitors of Keap1–Nrf2 PPI. Under the optimized conditions, the Keap1–Nrf2 TR-FRET assay exhibited great sensitivity with a high dynamic range and considerable stability for as long as 5 h. The Z’ factor was determined to be 0.82, suggesting that the assay is suitable for high-throughput screening and lead optimization of inhibitors of Keap1–Nrf2 PPI. Furthermore, the TR-FRET assay is capable of differentiating potent inhibitors of Keap1–Nrf2 PPI down to the subnanomolar inhibition constant ( Ki) range.

Time-Resolved Fluorescence Resonance Energy Transfer Shows that the Bacterial Multidrug ABC Half-Transporter BmrA Functions as a Homodimer†

Biochemistry ◽  
2005 ◽  
Vol 44 (11) ◽  
pp. 4312-4321 ◽  
Author(s):  
Olivier Dalmas ◽  
Marie-Ange Do Cao ◽  
Miguel R. Lugo ◽  
Frances J. Sharom ◽  
Attilio Di Pietro ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Fluorescence Resonance

Recommendations for the Reduction of Compound Artifacts in Time-Resolved Fluorescence Resonance Energy Transfer Assays

2007 ◽  
Vol 5 (3) ◽  
pp. 363-372 ◽  
Author(s):  
Pierre-Eloi Imbert ◽  
Vincent Unterreiner ◽  
Daniela Siebert ◽  
Hanspeter Gubler ◽  
Christian Parker ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Fluorescence Resonance
Sign in / Sign up

Export Citation Format

Share Document