Protein fluorophore labeling

GPI-anchored proteins (GP-APs) are synthesized in the cytoplasm and their transport into the ER occurs during the process of acquiring a GPI anchor, which is ultimately sorted into the outer leaflet of plasma membranes [24], A FRET study of fluorophore-labeled GPI-APs in cultured... 

The high molar absorptivities and quantum yields of the large protein fluorophore phycoerythrin (240,000 Da) have been exploited in energy transfer assays. Phyco-erythrin has been used as both donor and acceptor, with several bound antigen molecules per phycoerythrin molecule/86,94) The usefulness of BPE is indicated in competitive assays for human IgG that use fluorescein-labeled antibody as donor to... 

Transfer RNA (tRNA) molecules mediate translation of the nucleic acid genetic code into the amino acid building blocks of proteins, thus ensuring the survivability of cells. The dynamic properties of tRNA molecules are crucial to their functions in both activity and specificity. This chapter summarizes two methods that have been recently developed or improved upon previous protocols to introduce fluorophores to site-specific positions in tRNA. One method enables incorporation of fluorophores carrying a primary amine (such as proflavin or rhodamine) to dihydrouridine (D) residues in the tRNA tertiary core, and a second method enables incorporation of pyrroloC and 2-aminopurine to positions 75 and 76, respectively, of the CCA sequence at the 3 end. These site-specific fluorophore labeling methods utilize tRNA transcripts as the... 

Fluorescence polarization (anisotropy) version 2 (IMAP) Fluorophore labeled peptides bind to special detection beads coated with trivalent metal binding results in change in Brownian motion measured as with FP1 above Versatile without need for antibody Susceptible to compound interference peptide must be relatively small precludes use of protein substrates Turek-Etienne (2003a)... 

In some eases one of these two counterjarts is additionally labeled with a radionuclide, chromophore, fluorophore, enzyme, etc. to facilitate the detection and to make the method more sensitive. Leucoagglutinin labeled with tritium and protein A labeled with FITC can serve as examples. 

The proof-of-principle of the MAMEF technique, which couples the benefits of MEF with low power microwave heating to kinetically accelerate the bioassays, was first demonstrated with a model protein-fluorophore system,[l] with biotinylated-BSA and fluorophore-labeled streptavidin, as shown in Figure 7.3A. The... 

Three kinds of fluorescence probe were tested in the experimental system. They were (A) fluorophore labeled secondary antibody without GNP, (B) fluorophore labeled secondary antibody and GNP, which was suspended in PBS solution, and (C) fluorophore labeled secondary antibodies that are connected to protein A conjugated GNP (Au-PA). These are shown in Figure 8.26. Ten protein A molecules are bound to the surface of each GNP (266). Each protein A molecule contains four Fc terminal binding domains, and therefore, nearly 40 fluorophores on GNP are excited by the LSP field simultaneously. This arrangement is able to significantly enhance the intensity of fluorescence signal. 

Quantum yield added where available the quantum yield and fluorescent lifetimes can be highly dependent on the local environment. A fluorophore free in solution may have a different quantum yield than the same fluorophore attached to a protein, which in turn also depends on the extent of protein-to-fluorophore labeling (1-3). 

In contrast to common microarrays, the microsphere system uses optical methods to define the position and structure of a carbohydrate series [25], Incubation of the immobilized microsphere with a fluorophore-labeled carbohydrate-binding protein and the subsequent measuring of the fluorescence signals permits a determination of the binding profile. Binding events take place when one bead emits at both the wavelength of an internal code, which is used as a marker for the oligosaccharide attached to the microsphere, and the fluorophore-labeled protein. 

In an elegant approach toward DNA detection, Willner and colleagues introduced the use of catalytic beacons.Their method is illustrated in Figure 41. The thrombin protein was covalently modified with an oligonucleotide containing the TBA sequence.In the absence of a complementary DNA strand the appended TBA sequence folds into a G-quadruplex and blocks the enzyme s active site. Addition of a complementary DNA strand unfolds the G-quadruplex, resulting in substrate access to the thrombin s active site. Hydrolysis of a fluorophore-labeled peptide then results in a readily detectable optical signal. 

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