Fluorescently tagged molecules

Figure 6 Schematic depiction of a fluorescence anisotropy assay. Fluorescently tagged molecules are excited by plane-polarized light only molecules in the proper orientation are excited. Emitted light is detected in the original plane and in the perpendicular plane. The quantity of fluorescence observed in the two orientations is determined by the rate of tumbling, which depends on particle size and relates to binding.

In more modem methods, each of the dideoxy nucleotides carries a different fluorescent tag molecule, or fluorophor. Thus, all four chain-termination reactions can be carried out in a single sample tube. The different DNA strands are then separated by capillary electrophoresis and detected on the basis of their particular fluorescence. 

Physically distinct from FRAP and FRS is fluorescence correlation spectroscopy (FCS), in which optical methods are used to measure fluctuations in the number of fluorescently-tagged molecules in a small volume of space. In nondilute solution... 

Key words Optical patterning, Proteins, Photobleaching, Fluorescence-tagged molecules. Laser-... 

Dorn, 1. T., Neumaier, K. R. and Tampe, R. (1998) Molecular recognition of histidine-tagged molecules by metal-chelating lipids monitored by fluorescence energy transfer and correlation spectroscopy. ]. Am. Chem. Soc 120, 2753. 

Separation of amino acids, peptides, and proteins Amino acids are interesting molecules by themselves from an analytical point of view for two reasons. They are inherently enantiomeric and are the building blocks of peptides and proteins. The separation of amino acids is usually done through a derivatization process due to the fact that the absorbance in the UV is low. The most frequently used derivatization is done by fluorescent tagging. Sensitivity can reach the subfemtomole level.136 139 Temperature control can be used to separate conformers.140 Two conformers of Tyr-Pro-Phe-Asp-Val-Val-Gly-NH2 and four conformers of Tyr-Pro-Phe-Gly-Tyr-Pro-Ser-NH2 were separated at subzero temperatures by including glycerol as an antifreeze component of the buffer. 

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

Figure 5.27 SAED may be used to transfer the fluorescent AMCA label from the first molecule modified with the crosslinker to the second molecule crosslinked with it by reduction of its internal disulfide bond. Thus, unknown target molecules may be fluorescently tagged to follow them in vivo.

Fluorescent labels, by contrast, can provide tremendous sensitivity due to their property of discrete emission of light upon excitation. Proteins, nucleic acids, and other molecules can be labeled with fluorescent probes to provide highly receptive reagents for numerous in vitro assay procedures. For instance, fluorescently tagged antibodies can be used to probe cells and tissues for the presence of particular antigens, and then detected through the use of fluorescence microscopy techniques. Since each probe has its own fluorescence emission character, more... 

The level of TRITC modification in a macromolecule can be determined by measuring its absorbance at or near its characteristic absorption maximum ( 575nm). The number of fluor-ochrome molecules per molecule of protein is known as the F/P ratio. This value should be measured for all derivatives prepared with fluorescent tags. The ratio is especially important in predicting the behavior of antibodies labeled with TRITC. For a TRITC-labeled protein, the ratio of its absorbance at 575-280 nm should be between 0.3 and 0.7. 

In addition to the wide range of commercial probes, many other fluorescent molecules have been synthesized and described in the literature. Only a handful, however, are generally used to label antibody molecules. Perhaps the most common fluorescent tags with application to immunoglobulin assays are reflected in the main derivatives produced by the prominent antibody manufacturing companies. These include derivatives of cyanine dyes, fluorescein, rhod-amine, Texas red, aminomethylcoumarin (AMCA), and phycoerythrin. Figure 20.16 shows the reaction of fluorescein isothiocyanate (FITC), one of the most common fluorescent probes, with an antibody molecule. 

Purify the SH-labeled oligo by gel filtration on a desalting resin using 10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. The probe now may be used to conjugate with an activated enzyme, biotin, fluorescent tag, or other molecules containing a sulfhydryl-reactive group. 

FRET is a non-radiative transfer of energy from an excited donor fluorophore to an acceptor fluorophore which needs to be at a distance of-1-10 nm for conventional fluorescent tags (2-5) (see Fig. la). Energy transfer is characterized by the FRET efficiency E, which is the ratio of excited-state donor molecules relaxing by FRET to the total number of excited donors. The simplest way to... 

---