Labeling fluorescent dyes

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... 

Enzyme-labeled fluorescence (ELF) Various phosphate-labeled fluorescent dyes, ELF 97 Uses phosphatase-based signal amplification. Applicable to immunohisto-chemical and cytological staining, mRNA in situ hybridization, detection of endogenous phosphatase activity, and blot analyses. 34... 

In recent years, automated DNA sequencing machines capable of identifying about 10 bases per day have become commercially available. One clever innovation has been the use of fluorescent dyes of different colors to uniquely label the primer DNA introduced into the four sequencing reactions for example, red for the A reaction, blue for T, green for G, and yellow for C. Then, all four reaction mixtures can be combined and run together on one electrophoretic... 

I Very small amounts of the four 2, 3 -dicleoxyrtbonucleoside triphosphates (ddNTPs), each of which is labeled with a fluorescent dye of a different color (A 2 ,3 -d/ fco.Yyribonucleoside triphosphate is one in which both 2 and 3 -OH groups are missing from ribose.)... 

A diagnostic method using fluorescence labeled DNA probes to detect and quantify the number complementary chromosomal sequences on a cellular resolution. A related technique that also allows assessment of gene amplifications, but without precise quantification of copy numbers is the chromogenic in situ hybridization (CISH). Here, instead of a fluorescent dye an enzyme that can generate a colored precipitate in the tissue samples is coupled to the DNA probe. 

A relatively large amount of RNA needs to be labeled with fluorescent dyes in order to obtain satisfactoiy results (20-100 pg of total RNA or 1-2 pg of mRNA). These requirements could make microarrays incompatible with the very limited RNA yields obtained from some... 

Inoculation of cell cultures with virus-containing material produces characteristic changes in the cells. The replication of many types of viruses produces the cytopathic effect (CPE) in which cells degenerate. This effect is seen as the shrinkage or sometimes ballooning of cells and the disruption of the monolayer by death and detachment of the cells (Fig. 3.6). The replicating virus can then be identified by inoculating a series of cell cultures with mixtures of the virus and different known viral antisera. If the virus is the same as one of the types used to prepare the various antisera, then its activity will be neutralized by that particular antiserum and CPE will not be apparent in that tube. Alternatively viral antisera labelled with a fluorescent dye can be used to identify the virus in the cell culture. 

Direct observation of molecular diffusion is the most powerful approach to evaluate the bilayer fluidity and molecular diffusivity. Recent advances in optics and CCD devices enable us to detect and track the diffusive motion of a single molecule with an optical microscope. Usually, a fluorescent dye, gold nanoparticle, or fluorescent microsphere is used to label the target molecule in order to visualize it in the microscope [31-33]. By tracking the diffusive motion of the labeled-molecule in an artificial lipid bilayer, random Brownian motion was clearly observed (Figure 13.3) [31]. As already mentioned, the artificial lipid bilayer can be treated as a two-dimensional fluid. Thus, an analysis for a two-dimensional random walk can be applied. Each trajectory observed on the microscope is then numerically analyzed by a simple relationship between the displacement, r, and time interval, T,... 

NOTE Retrogradely labeled cells were counted after a fluorescent dye was injected in... 

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. 

A variation on this method, called fluorescent in situ hybridization (FISH), uses fluorescent-labeled DNA and RNA probes for detection and visualization of single cells by microscopy or flow cytometry.7 80 The FISH technique is popular because of its sensitivity and speed of visualization fluorescent dyes can be used to produce probes with different colors for simultaneous detection of several organisms.76,81,82... 

The NIR fluorescent 2,3-dihydroperimidine-squaraines 35a,b were synthesized and used as biological labels [99]. Dye 35b including four sulfopropyl groups exhibits high water-solubility, an emission maximum at 812 nm (protein ratio D/P = 1), and 817 nm in the presence of BSA, which indicates compatibility with commercially available NIR laser diodes. 

Due to their low sensitivity toward the environment, cyanine dyes are perfect candidates as fluorescent labels. Squaraine dyes on the other hand display a highly environment-sensitive response and are therefore not only useful as fluorescent probes and labels but also, in particular, well-suited for lifetime-based applications. 

In an indirect comparison design, the free and polysome samples are labeled with a red fluorescent dye, and each is hybridized independently to a DNA microarray against an RNA sample that is labeled with green fluorescent dye (Fig. 10.IB). Because the green-labeled sample is the same for both samples, it serves as a common reference and the polysome-to-free ratio can... 

Organic fluorescent dyes with the appropriate spectral properties also can be paired with lanthanide chelates in FRET systems. For instance, many rhodamine dyes and the cyanine dye Cy5 have ideal excitation wavelengths for receiving energy from a nearby europium chelate. The LeadSeeker assay system from GE Healthcare incorporates various Cy5-labeled antibodies for developing specific analyte assays. In addition, if using a terbium chelate as the donor, then a Cy3 fluorescent dye can be used in assays as the acceptor. 

In a DNA array, gene-specific probes are created and immobilized on a chip (silicon wafer, nylon or glass array substrate). Biological samples are labeled with fluorescent dyes or radioactivity. These labeled samples are then incubated with the probes to allow hybridizations to take place in a high fidelity manner. After incubation, non-hybridized samples are washed away and spot fluorescent or radioactivity signals resulting from hybridization can be detected. 

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