Fluorescent dye-binding

Fig. 33.2. The CeneChip system RNAs isolated from herbicide treated plants and labeled with a fluorescent dye bind to their corresponding gene probes. Highly abundant RNAs produce bright signals, whereas rare RNAs produce only dim signals.

Fluorescent dye-binding 1 ng to 10 fig Varies with dye Depends (often no) 0.2-2.5 mL... 

Several other techniques for have evolved for biochemical assays. In chapter 2 of this book, Omann and Sklar report on a method of fluoroimmunoassay where the bound and unbound antigen are separated by the quenching of fluorescence that accompanies antibody binding. Then, in chapter 3, Holl and Webb show how they achieved a sensitive measurement of nucleic acids by the enhancement in fluorescence that accompanies the binding of fluorescent dyes to nucleic acids. Chandler et al, also used fluorescence enhancement to monitor calcium mobility in neutrophil cells. 

The theory and application of this fluorescence method have been discussed in detail by LePecq and others (3,8). The assay requires that there is sufficient ionic strength to minimize ionic binding (e.g., O.IM sodium chloride), that the pH is 4-10, that no heavy metals are present, that the fluorescence is not enhanced on binding to other excipients (e.g., proteins) and that at least portions of the nucleic acids are not complexed. These requirements can usually he met when dealing with recombinant products in some cases the samples must he manipulated to create the appropriate conditions. In the intercalative method of dye binding, proteins rarely interfere with the assay, and procedures have been developed to remove the few interferences they may cause (e.g., the use of heparin or enzymatic digestion of the protein 9). 

The LANCE cAMP assay is a competitive assay in which cAMP produced by the cells competes with fluorescent-labeled acceptor cAMP for a cryptate tagged donor antibody. The principal of the assay is shown in Fig. 6. On the left strepta-vidin conjugated Europium binds to biotinylated cAMP. An antibody labeled with the fluorescent dye Alexa binds to the cAMP, bringing the donor and acceptor into close proximity, and energy transfer occurs. When the cell releases cAMP, it competes with the biotin-labeled cAMP for the antibody, and a signal decrease is observed. In the TR-FRET assay the antibody is directly labeled with either Eu or Tb. In this format an increase in cAMP also causes a decrease in signal. 

This dye fluoresces after binding Pb+2 and Ca+2 lead is considered an interferant to the determination of calcium by this approach. However, by complexing the divalent lead ion with the heavy metal chelator TPEN (N,N,N ,N -tetrakis(2-pyridylmethyl)ethylene-diamine) prior to the addition of the fluo-3, the fluorescent... 

Black, S. L., Stanley, W. A., Filipp, F. V., Bhairo, M., Verma, A., Wichmann, O., Sattler, M., Wilmanns, M. and Schultz, C. (2008). Probing lipid- and drug-binding domains with fluorescent dyes. Bioorg. Med. Chem. 16, 1162-1173. 

Fluorescence occurs when radiant energy is absorbed and then, almost instantly, some of the energy is re-emitted, usually at a longer wavelength. Primary fluorescence (autofluorescence) occurs in flavo-proteins (13), plant cell wall materials such as lignin (7), and in flagella (14). Secondary fluorescence is when a material binds a fluorescent dye... 

In summary, the encapsulation of cyanine dyes in CB7 causes either an increase or a decrease in quantum yields and brightness but in general increases photostability. Enhancements in fluorescence intensity by about one order of magnitude or more were observed [46]. These fluorescence property changes are utilized for the development of sensors, where the fluorescent dye may serve as a probe to signal the binding of an analyte. No reports were found on the encapsulation of squaraines in CBs. 

DNA is often present in amounts too small to be detected by direct spectroscopy. In this case, the fluorescent dye EtBr can be used to amplify the absorption. EtBr binds to the DNA molecule by intercalating between adjacent base pairs. It absorbs ultraviolet light at 300 nm and emits light at 590 nm in the red/orange region of the visible spectrum. The method can be used to determine the amount of DNA in a test-tube by comparing the EtBr-mediated fluorescence of the sample with that of standards of known amounts of DNA. 

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