A comparative analysis of resonance energy transfer methods for Alzheimer related protein-protein interactions in living cells
| Autor: | Honggun Lee, Doyoon Kwon, Regis Grailhe, Joohyun Lee, Jiho Kim |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
Fluorescence-lifetime imaging microscopy
Nerve Tissue Proteins Protein–protein interaction Amyloid beta-Protein Precursor Amyloid precursor protein Fluorescence Resonance Energy Transfer Humans Binding site Molecular Biology Adaptor Proteins Signal Transducing Binding Sites biology Chemistry HEK 293 cells Signal transducing adaptor protein Nuclear Proteins Flow Cytometry Molecular biology Förster resonance energy transfer HEK293 Cells Energy Transfer Microscopy Fluorescence Luminescent Measurements Biophysics biology.protein Cytometry Biotechnology |
| Zdroj: | Molecular bioSystems. 7(11) |
| ISSN: | 1742-2051 |
| Popis: | Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) are extensively used to analyze protein interactions occurring in living cells. Although these two techniques are broadly applied in cellular biology, comparative analysis of their strengths and limitations is lacking. To this end, we analyzed a small network of proteins involved in the amyloidogenic processing of the Alzheimer β-amyloid precursor using FRET based cytometry, BRET, and fluorescence lifetime imaging microscopy (FLIM). Using all three methods, we were able to detect the interactions of the amyloid precursor protein with APBB1, APBB2, and APP itself. And we found an unreported interacting pair, APP-APH1A. In addition, we show that these four interacting pairs exhibit a strong FRET correlation with the acceptor/donor expression ratios. Overall the FRET based cytometry was the most sensitive and reliable approach to screen for new interacting proteins. Therefore, we applied FRET based cytometry to study competitive binding of two proteins, APBB1 and APBB2, with the same APP target. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|