Labeling, labels fluorescence

Ino H. Application of antigen retrieval by heating for double-label fluorescent immunohistochemistry with identical species-derived primary antibodies. J. Histochem. Cytochem. 2004 52 1219-1230. 

Thus, this reagent can be used to label fluorescently proteins and other biomolecules containing free sulfhydryl residues. If there are no —SH groups available, their creation can be accomplished by reduction of indigenous disulfides or through the use of various thiolation reagents (Chapter 1, Section 4.1). 

Synthesis of tritium-labelled fluorescent derivatives of prostaglandins... 

For both organic dyes and QDs, bioconjugation often leads to a decrease in fluorescence quantum yield and thus typically also in emission lifetime. Parameters that can affect label fluorescence are the chemical nature and the length of the spacer and, at least for organic dyes, the type of neighboring biomolecules like oligonucleotides or amino acids in the bioconjugated form. 

The hydrolysis of a nonfluorescent enzyme substrate to a fluorescent product is widely utilized to measure the activity of a large number of enzymes. Binding of enzyme substrates by antibodies often protects the enzymatically labile bond from hydrolysis. By the combination of these two formats, the substrate-labeled fluorescent immunoassay (SLFIA) was developed. ... 

Figure 6.2. Chemical structure of 8-[3-(7-/)-galactosylcoumarin-3-carboxyamido )propyl ] theophylline (B) used in the homogeneous substrate-labeled fluorescent immunoassay for theophylline (A). (Reprinted from Ref. 3, with permission from the American Association for Clinical Chemistry.)...

In the past ten years, numerous applications of fluorescence methods for monitoring homogeneous and heterogeneous immunoassays have been reported. Advances in the design of fluorescent labels have prompted the development of various fluorescent immunoassay schemes such as the substrate-labeled fluorescent immunoassay and the fluorescence excitation transfer immunoassay. As sophisticated fluorescence instrumentation for lifetime measurement became available, the phase-resolved and time-resolved fluorescent immunoassays have also developed. With the current emphasis on satellite and physician s office testing, future innovations in fluorescence immunoassay development will be expected to center on the simplification of assay protocol and the development of solid-state miniaturized fluorescence readers for on-site testing. 

Note ELF = enzyme labeled fluorescence. ECL = enhanced chemiluninescence. CL = chemiluminescence. RES = biotin amplification, sAV-Cy3. RCA = rolling circle amplification. 

Tyramide signal amplification (TSA PerkinElmer Life Sciences, Boston) and enzyme-labeled fluorescence (ELF Molecular Probes) are related detection technologies. In the tyramide amplification process, a tyramide-biotin complex is produced by the action of horseradish peroxidase. The complex precipitates near the binding site and accumulates. The complex is detected by the use of streptavidin-Cy3/Cy5. 

Figure 6.24 Enzyme-labeled fluorescence. ELF-97 is a soluble phosphorylated substrate cleaved by alkaline phosphatase into a highly fluorescent, insoluble product. (Molecular Probes, Inc., Eugene, OR.)...

A useful approach to monitor microbial populations in the biotreatment of hazardous wastes involves the detection of specific sequences of nucleic acids by hybridization with complementary oligonucleotide probes. Radioactive labels, fluorescent labels, and other kinds of labels are attached to the probes to increase sensitivity and simplicity of the hybridization... 

Biotinylated p53 peptide with fluorescence label Fluorescence polarization... 

Methods for the Detection of Antigens/Antibodies Equilibrium and kinetic inhibition assays based upon fluorescence polarization, 70, 3 fluorescence excitation transfer immunoassay (FETI), 70, 28 indirect quenching fluoroimmunoassay, 70, 60 the homogeneous substrate-labeled fluorescent immunoassay, 70, 79 fluorescence immunoassays using plane surface solid phases (FIAPS), 70, 87. 

Examples of its use include protein and nucleic acid detection, enzyme-labelled fluorescence, in the intrinsic fluorescence of normal and cancer cells, as external... 

Fluorescein-labeled proteins are also used to measure the translational mobility of proteins and lipids by the Fluorescence Recovery After Photo-bleaching technique [54-59]. The uniformly labeled fluorescent sample is flashed with an intense light source to bleach a spot, thus producing a concentration gradient. The rate of recovery of fluorescence in that bleached area is measured and used to calculate the diffusion coefficient of the probe dye into the bleached zone. Such diffusion coefficient measurements have been used to determine the association constants of proteins in cells [60], to measure the exchange of tubulin between the cytoplasm and the microtubules [61,62], to study the polymerization-depolymerization process of actin [63-65] and to monitor the changes that occur upon cell maturation [66,67]. 

Hydrazide groups directly react with aldehyde and ketone functional groups to form relatively stable hydrazone linkages (Chapter 2, Section 5.1). Two fluorescein derivatives are commonly available that contain hydrazide groups off their No. 5 carbons on the lower ring structure. Both may be used to label fluorescently aldehyde- or ketone-... 

Workers with James Jett at Los Alamos have been developing cytometers that are able to detect the fluorescence from single molecules and have been applying this capability to the problem of DNA sequencing. The proposed technique involves the synthesis of a complementary strand of DNA with fluorescently tagged precursors (each base of a different color). The labeled (fluorescent) duplex DNA molecule is then attached to a microsphere and the microsphere suspended in a flow stream where it can be sequentially cleaved with an exonuclease. The cleaved fluorescent bases from the molecule would then be identified by their color as they pass through the laser beam, and the sequence in which the colors appear will reflect the base sequence in the DNA. It is projected that between 100 and 1000 bases per second could be sequenced, although there are still obstacles to be surmounted before this technique becomes a practical reality. 

A four-layer microchip has been constructed to generate total internal reflection (TIR) and an evanescent field (see Figure 7.8). Surface-adhered Nile red-labeled fluorescent microspheres (1 pm) are excited by the evanescent field for fluorescent measurement. An essential feature on the chip was the micromirror that was constructed by depositing Au/Cr on the slanted wall (54.7° due to anisotropic etch of Si). Operation near the critical angle 0C assures strong evanescent intensity [695]. 

Fig. 4. Schematic representation of the principle of substrate-labeled fluorescent immunoassay (tda, Ames). [Cited and modified from Fig. B-5, Miyai, K., in Miyai etal., eds. (M10).]...

B6. Burd, J. F., Wong, R. C., Feeney, J. E., Carrico, R. J., and Boguslaski, R., Homogeneous reactant-labeled fluorescent immunoassay for therapeutic drugs exemplified by gentamicin determination in human serum. Clin. Chem. 23, 1402-1408 (1977). 

The importance of proper instrument and experimental standards to evaluate the machine performance and experimental variables should not be underestimated. We recommend to check for even illumination, PSF, system/laser stability and correct deviations, when necessary, using proper procedures such as fluorescence slides and triple or double-labeled fluorescence beads containing defined amounts of fluorophores. 

The second group consist of the real fluorescent labels (fluorescent dyes) which are characterized by forming covalent bonds with the labeled protein. The third and... 

Cotton, G. J. and Muir, T. W. (2000) Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation. Chem. Biol. 7, 253-261. 

Randolph, J.B. and Waggoner, A.S. (1997) Stability, specificity and fluorescence brightness of multiply-labelled fluorescent DNA probes. Nucleic Acids Res 25 2923-9. 

Improving specificity of Binary aptamer Noncovalent labeling Fluorescent sensing 37... 

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