Tags, fluorescent

This group was developed as part of a scheme to prepare fluorescent tags to be used in DNA sequencing. Deprotection is accomplished by irradiation at 360 nm to release the NVOC group, which then sets up the system to form a diketopiper-azine while releasing the alcohol. ... 

We have also used a non-radiometric-binding approach based on fluorescence polarisation [29], where a fluorescent label is used in place of a radiolabel. As the fluorescently tagged oxytocin binds to the receptor, its rotational velocity is reduced and the polarisation of the fluorophore increases. The displacement of the ligand may be measured by a decrease in polarisation. 

Another popular assay format for kinase assays is the Lanthascreen. This format is a variation on the LANCE assay, but employs Tb as the cryptate. In this format N-terminally fluorescently tagged peptide substrate (acceptor) is phosphorylated by the kinase. Next, a phophospecific antibody which is labeled with terbium binds specifically to the phosphorylated product, placing the donor and acceptor in close proximity, generating a signal [25]. 

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. 

Figure 7.1. Protein expression mapping using an antibody array. The antibody array consists of monoclonal antibodies specific for a set of proteins in the organism of interest gridded onto a filter. To determine if a protein is expressed under the conditions being tested, a crude lysate is obtained and the proteins within the lysate are labeled with a fluorescent tag. The lysate is applied to the filter and the proteins are allowed to bind to the relevant antibody. Bound proteins are visualized via the fluorescent tag.

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... 

Second, the technology has mediocre reproducibility. Software is available to morph images so that spots can be lined up such software is expensive, difficult to use, and not always accurate in its alignment. To overcome this problem and to simplify quantitative comparisons between samples, Unlu et al. (1997) developed differential gel electrophoresis (DIGE), where two samples are each labeled with different fluorescent tags, pooled, separated on the same gel, and scanned at characteristic wavelengths to resolve the components. This technology has been commercialized by Amersham. 

Recently, Beatty and Tirrell [201] relied on the simultaneous or sequential addition of two reactive Met analogs, Aha and Hpg, to enable the fluorescent tagging of two protein populations within cells. The first demonstration of two-dye labeling of metabolically tagged cells was described in 2007 by Chang and co-workers [202], who used flow cytometry to show that cells treated with two reactive sugars could be labeled with distinct fluorophores. 

Simard JR, Getlik M, Griitter C et al (2009) Development of a fluorescent-tagged kinase assay system for the detection and characterization of allosteric kinase inhibitors. J Am ChemSoc 131 13286-13296... 

Faller T, Hutton K, Okafo G, et al (1997) A novel acridone derivative for the fluorescence tagging and mass spectrometric sequencing of peptides. Chem Commun 1529-1530... 

Kottegoda S, Aoto PC, Sims CE, Allbritton NL (2008) Biarsenical—tetracysteine motif as a fluorescent tag for detection in capillary electrophoresis. Anal Chem 80 5358-5366... 

Therefore, a good catalyst leads to weak fluorescence in the Heck product (15). A relatively small library of 96 known phosphines was tested in the palladium-catalyzed Heck coupling. Several sterically hindered ligands led to high catalyst activity. Fluorescence tags have also been used in the combinatorial search for metal-free catalysts in other types of reaction.42,43... 

Lauf, U., Lopez, P. and Falk, M. M. (2001). Expression of fluorescently tagged connexins A novel approach to rescue function of oligomeric DsRed-tagged proteins. FEBS Lett. 498, 11-5. 

Excellent protocols for transient expression of fluorescently tagged proteins in N. benthamiana leaves using Agrobacterium-mediated infiltration are available [99, 100],... 

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. 

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