Fluorophores terminal labeling

Figure 8.2 Orientation of transition moments of cyanine fluorophores terminally attached to double-stranded DNA. (A) The orientation parameter K2. The transition dipole vectors for the coupled donor and acceptor fluorophore are indicated by the arrows, labeled D and A. Vector A is generated by the in-plane translation of vector A to share its origin with vector D. The definition of K2, given in Eq. (8.5), is based upon the angles shown. (B) If the fluorophores he in parallel planes, the orientation parameter simplifies to K2 — cos2 T and varies between 0 and 1. The schematic shows the limiting cases, where the transition moments are parallel (k2 = 1) and crossed (K2 — 0). If the transition moments are colinear, K2 = 4.

Another group has recently performed PBM experiments using the N-terminal domain of the Drosophila TF Extradenticle directly labeled with the fluorophore Cy3 at a unique cysteine [29]. Yet another group, using directly labeled TFs for binding to dsDNA microarrays, found that the TF Jim C-terminally labeled with Cy5-dC-puromycin was capable of interacting with... 

DNA modified with a diamine compound to contain terminal primary amines may be coupled with amine-reactive fluorescent labels. The most common fluorophores used for oligonucleotide labeling are the cyanine dyes and derivatives of fluorescein and rhodamine (Chapter 9). However, any of the amine-reactive labels discussed throughout Chapter 9 are valid candidates for DNA applications. 

The N-terminal amino group or a Lys residue can easily be used to label the PNA. This can be carried out in solution after purification, or more conveniently, while the PNA is still attached to the resin. Fluorescein and rhodamine are the most common fluorophore labels, while biotin is the most common affinity tag. Fluorescein or rhodamine are usually coupled to the amino group as -OSu esters or isothiocyanates. 4,4 -Dimethoxytrityl-protected biotin and Piv-pro-tected fluorescein have also been coupled to the N-terminus of a PNA as their 1-phenylpyr-azolin-5-one carboxylate esters.147 In 1997 a new fluorescein-conjugated Lys monomer (21, Scheme 12) was described. 48 ... 

DNA modified with a diamine compound to contain terminal primary amines may be coupled with amine-reactive fluorescent labels. The most common fluorophores used... 

FIGURE 2.2 Fluorescence intensity readout principle. In the intact peptidic substrate (amino acids symbolized by X, Y, and Z) labeled with a fluorophore at the C terminus, the intensity of fluorescence emission (light gray arrow) after excitation (dark gray arrow) is low. Through the cleavage of the substrate between the C terminal amino acid (Z) and the fluorophore by a protease, the intensity of fluorescence emission is strongly enhanced. An increase of fluorescence intensity over time dependent on the enzymatic velocity is observed. 

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. 

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