GFP - Imaging Fluorescent Proteins

Applications

XFPs as passive Indicators

Monitoring the appearance, degradation, protein turnover, location or translocation of your protein of interest with the help of an XFP protein partner.

XFP as Marker of Gene Expression

• as a reporter gene for gene expression located behind the promoter of interest. Prerequisite are stably transfected cells or transgenic organisms. Method replaces enzymatic expression reporting systems base on β-galactosidase or β-lactamase.
• XFP as a marker in cotransfection in transienty transfected cells: transfected cell can be identified quickly before the experiment (not through an antibody stain afterwards), course of transfection (expression levels) can be followed easily

XFP-fusion proteins to image protein of interest in living cells in real time

• Spatial marker for proteins in living cells: allows precise location of the protein of interest without the risk of artefacts due to fixation and/or staining procedures
• XFP as a tool to visualize intracellular organelles (or other subcellular structures) in living cells (multi-labeling):
  Clontech provides vectors targeted to: actin, tubulin, mitochondria, nucleus, endoplasmic reticulum, Golgi, PM, preoxisomes, endosomes (early, late): colocalization with protein of interest can be detected easily
• protein/organell dynamic (multiple proteins with different XFPs) in real time
• Fluorescence speckle microscopy (8): monitoring cytoskeletal dynamics
• localization of intracellular messenger molecules (3-PIs, DAG & PA) (9, 10, 11)
• RNA localization (67-70)
• gene activity and change in chromatin structure (71-74)
• protein dynamics with temperature-sensitive GFP-mutants (12, 13): pulse-chase type experiments
• studies of protein dynamics with bleaching experiments:
  FRAP = fluorescence recovery after photobleaching (ref.)
  FLIP = fluorescence loss in photobleaching (ref.)

XFPs as active Indicators

Quantitative measurements using changes in the fluorescence spectra/intensity of XFPs

• pH-measurements with engineered GFPs (14, 15)
• Cl^- fluxes with halide-sensitive YFPs (16)
• Calcium-Sensors:
  • Camgaroos: Calmodulin inserted into YFP => fluorescence intensity increases sevenfold on binding of Ca²⁺ . Used to measure Ca²⁺ -concentration inside Mitochondria (17).
  • Pericams (18): circularly permuted GFP (N- and C-termini connected by flexible linker, new N- and C- termini introduced close to chromophore: higher sensitivity of fluorescence towards mechanical stresses) is inserted between calmodulin and M13 to yield Ca²⁺ sensor. More sensitive than Camgaroos, some Pericams increase wavelength, some shift excitation wavelength: ratiometric dyes (Chroma filter set 71013)
• XFP-Sensors based on intramolecular FRET for
  • Ca²⁺ (Cameleons)
  • Indicators of protease activity
  • other cellular parameters:
    - cGMP
    - phosphorylation
    - membrane potential
    
• XFP-sensors based on intermolecular FRET
  • Indicators for cAMP
  • Protein-Protein-Interaction:
    - Description factors
    - G-proteins
    

For details on FRET see Applications