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Protein Interactions and Proteomics Facility Core Fluorescence Resonance Energy Transfer (FRET) 1. Description: FRET measures the non-radioactive transfer of energy from one fluorescent center to another. The efficiency of the transfer is proportional to the distance between the two fluorescent centers raised to the 6th power. Therefore, FRET is used to demonstrate that two fluorescent molecules are in close proximity and in some instances can be used to estimate the distance between them. Biosensors are a subset of FRET experiments in which the two fluorescent centers are constructed to be in the same molecule and are separated by a “tether’ that is sensitive to a signaling molecule. When a signaling molecule is introduced the centers either bind to each other or are released resulting in a change in the FRET between them. Biosensors exist for Ca2+, calmodulin, cAMP, kinases etc. 2. Application: Detection of protein interactions inside living cells Characterization of binding between fluorescent species in vitro Detection of signaling events in cells when a ‘biosensor” is available 3. Limitations: The two fluorescent species must be introduced in the cell at appropriate concentrations The number of technically useful FRET pairs is very limited. The best is CFP/YFP and the overlap of their spectra means that the signal must be computationally adjusted to measure FRET. The limits of quantum efficiency and photo bleaching that are standard for all fluorescence work 4. Advantages: This is the best technique available to monitor protein binding and signaling events in real time in live cells Biosensor probes can be designed to localize in subcellular compartments such as the ER or Nucleus 5. Equipment: Zeiss Axiovert Inverted Fluorescent Microscope: This microscope is equipped will all hardware and software for analysis of FRET and can monitor fluorescent signals over time using repeated measures of identified cells. QM-6 Spectrofluorometer: This is a monochrometer based dual emission spectrofluorometer. It is used for in vitro FRET experiments and to verify the behavior of FRET pairs before they are used in cells. 6. Services: Consultation on FRET Training in the use of the Zeiss microscope and QM-6 spectrofluorometer Use of cell lines that have biosensors established in them for both training and experimental purposes Use of cDNA constructs within the core so the Investigators gene of interest can be inserted into a constructs for expression of a fluorescent fusion proteins |
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