Organelle-specific fluorescent labels

Instead of performing time-consuming and tedious organelle purification techniques, it is often possible to use organelle-specific fluorescent labels to provide the necessary detection selectivity. Such labels allow crudely purified organelle fi actions, prepared from differential centrifugation, to be analyzed. Many organelle-specific fluorescent labels are commercially available and Table 20.3 lists some of the most commonly used labels. 

In immunofluorescence microscopy, specific proteins and organelles in fixed cells are stained with fluorescence-labeled monoclonal antibodies. Multiple proteins can be localized in the same sample by staining with antibodies labeled with different fluorochromes. 

In the biological chemistry field, fusion proteins have recently shown more potential as organelle-specific labels. For subcellular analysis, fusion proteins can be expressed that contain both a fluorescent protein and a subcellular localization sequence. This combination results in organelle-specific labeling that retains the excellent photochemical properties of fluorescent proteins (e.g., intense fluorescence and photostability). We commonly use a commercially available plasmid that codes for a fusion protein of red fluorescent protein (DsRed2) and the mitochondrial targeting sequence from subunit VIII of cytochrome c oxidase, which contains the neomycin/kanamycin resistance gene, as described below. 

A number of approaches to fluorescently label cytoplasmic and nuclear structures or organelles in living cells are presently available. They vary considerably in terms of ease of use, how specifically they label the target molecules, and in how much they interfere with cellular function. Direct microinjection of fluorescently labelled antibodies into living cells is a universal and efficient way of labelling cellular targets in vivo. In order to avoid interference with cellular function or non-specific cross-reactions the fluorescent antibodies should be prepared as described in Section 2.1 and be first characterized in vitro before they are introduced into cells. 

Green fluorescent protein (GFP) and related fluorescent proteins can be used to label practically any protein or subcellular compartment of living cells (49). Transfection of cells with plasmids that encode appropriately targeted fluorescent fusion proteins has been used to define the plasma membrane, early endosomes, late endosomes, caveolae, the golgi complex, the ER, and other subcellular locations. Several fluorescent small molecules are also available for labehng specific cellular organelles, including endosomes and lysosomes, for analysis by fluorescence microscopy. 

Fluorescein tetramethylrhodamine dextran (FRD) was used as a ratiometric probe, which provides a pH-independent signal (tetramethylrhodamine), to compensate for different organelle volumes, and a pH-dependent signal (fluorescein). The ratio of the pH-independent and pH-dependent fluorescence was expected to provide a quantitative measure of the pH. Acidic organelles (lysosomes and endosomes) were specifically labeled by FRD, because the cells endocytose small amounts of the extracellular medium, which is accumulated in endosomes and eventually lysosomes. CE was performed in a poly((V-acryloyl aminopropanol, AAP) modified capillary to reduce both adsorption at the capillary surface and the electroosmotic flow. LIF detection was performed off-column using a... 

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