Using Organic Fluorescent Probes in Combination with GFP—Note 12.1

The Green Fluorescent Protein (GFP) reporter has added a new dimension to the analysis of protein localization, allowing real-time examination in live cells of processes that have conventionally been observed through immunocytochemical "snapshots" in fixed specimens. Using spectrally distinct, organic fluorescent probes and markers (Table 1) adds extra data dimensions and reference points to these experiments (Figure 1).

Figure 1. The morphology of sporulating Bacillus subtilis in the early stages of forespore engulfment. The membranes and chromosomes of both the forespore and the larger mother cell are stained with FM 4-64 (red; T3166, T13320) and DAPI (blue; D1306, D3571, D21490), respectively. The small green-fluorescent patch indicates the localization of a GFP fusion to SPoIIIE, a protein essential for translocation of the forespore chromosome that may also regulate membrane fusion events (see Proc Natl Acad Sci U S A (1999) 96:14553). The background contains sporangia at various stages in the engulfment process stained with MitoTracker Green FM (green, M7514) and FM 4-64 (red).

The majority of the applications summarized in Table 1 involve live cells, tissues and organisms. There are many other instances where research objectives call for complementary use of immunochemical and GFP-based protein localization techniques. These experiments demand the combination of brightness, photostability and spectral separation provided by our Alexa Fluor dye–labeled secondary detection reagents. For two-color combinations with GFP, we recommend the Alexa Fluor 555, Alexa Fluor 568 or Alexa Fluor 594 dye–labeled secondary antibodies (Secondary Immunoreagents—Section 7.2, Summary of Molecular Probes secondary antibody conjugates—Table 7.1). The addition of Alexa Fluor 635 or Alexa Fluor 647 dye–labeled antibodies allows three-color detection. Some immunohistochemical procedures such as paraffin embedding of fixed tissue result in loss of the intrinsic fluorescence of GFP. In other cases, GFP expression levels may simply be too low for detection above background autofluorescence. Antibodies to GFP provide remedies for these problems (Figure 2). We offer unlabeled mouse and rabbit monoclonal GFP antibodies and unlabeled rabbit and chicken polyclonal GFP antibodies, as well as Alexa Fluor dye–labeled rabbit polyclonal GFP antibodies (Antibodies against Expression Tags—Section 7.5).


Figure 2. HeLa cell transfected with pShooter pCMV/myc/mito/GFP, then fixed and permeabilized. Green Fluorescent Protein (GFP) localized in the mitochondria was labeled with mouse IgG2a anti-GFP antibody (A11120) and detected with orange-fluorescent Alexa Fluor 555 goat anti–mouse IgG antibody (A21422), which colocalized with the dim GFP fluorescence. F-actin was labeled with green-fluorescent Alexa Fluor 488 phalloidin (A12379), and the nucleus was stained with blue-fluorescent DAPI (D1306, D3571, D21490). The sample was mounted using ProLong Gold antifade reagent (P36930). Some GFP fluorescence is retained in the mitochondria after fixation (left), but immunolabeling and detection greatly improve visualization (right).

Proximity-dependent fluorescence resonance energy transfer (FRET) allows detection of protein–protein interactions with much higher spatial resolution than conventional diffraction-limited microscopy. Alexa Fluor dyes with strong absorption in the 500–600 nm wavelength range are excellent FRET acceptors from GFP (Table 2). An assay to detect activation of GFP–GTPase fusions developed by researchers at Scripps Research Institute utilizes the GTPase-binding domain (PBD) of PAK1, a protein that binds to GTPases only in their activated GTP-bound form. GTPase activation is indicated by FRET from GFP to PDB labeled with Alexa Fluor 546 C₅-maleimide at a single N-terminal cysteine residue. This assay has been used to determine the location and dynamics of rac and Cdc42 GTPase activation in live cells.

In 2002, researchers in Scott Fraser's laboratory at the California Institute of Technology reported a method of coinjecting Texas Red dye–labeled 10,000 MW dextran and GFP vectors into sea urchin embryos. This method overcomes a multitude of problems inherent in making intra- and inter-embryo comparisons of gene expression levels using confocal microscopy. In particular, laser excitation and fluorescence collection efficiencies vary with the depth of the fluorescent protein in the embryo, and the orientation of different embryos on the coverslip varies relative to the microscope objective. Measuring the ratio of GFP fluorescence to Texas Red dextran fluorescence corrects for these spatial factors, providing a gene expression readout that is 2–50 times more accurate than conventional confocal microscopy procedures depending on the localization of GFP within an embryo. A similar strategy was previously used to determine translation efficiencies of GFP-encoding mRNAs.