Tetramethyl rhodamine fluorescent

Fluorescence detection relies on the visualization of a secondary antibody that has been labeled with a fluorophore such as fluorescein (FITC), Texas Red, Tetramethyl rhodamine (TRITC), or R-phycoerythrin. Although this method of detection has a reduced sensitivity of twofold to fourfold compared to chemiluminescence detection, it presents a tenfold greater linear dynamic range, thus providing better linearity and better quantiflcation within the detection limits. Since secondary antibodies can be labeled with fluor-ophores of distinct colors, multiplexing (simultaneous detection of several antigens) of the same blot is feasible. 

Fluorescence resonance energy transfer (FRET) is a technique that has been used to measure distances between pairs of proximal fluorochromes. A suitable pair consists of a donor fluorochrome, which has an emission spectrum that significantly overlaps with the absorption spectrum of an acceptor fluorochrome (2). With the availability of monoclonal antibodies to many cell-surface determinants, intramolecular distances between nearby epitopes and intermolecular distances between adjacent cell-surface macromolecules can be investigated to analyze molecular interactions influencing important cellular events. Such monoclonal antibodies can be conjugated to fluorescein-isothiocyanate (FITC) as the donor, and either tetramethyl-rhodamine-isothiocyanate (TRITC) or phycoerythrin (PE) as the acceptor. 

Fig. 24.2. Single-molecule recording of T4 lysozyme conformational motions and enzymatic reaction turnovers of hydrolysis of an E. coli B cell wall in real time, (a) This panel shows a pair of trajectories from a fluorescence donor tetramethyl-rhodamine blue) and acceptor Texas Red (red) pair in a single-T4 lysozyme in the presence of E. coli cells of 2.5mg/mL at pH 7.2 buffer. Anticorrelated fluctuation features are evident. (b) The correlation functions (C (t)) of donor ( A/a (0) Aid (f)), blue), acceptor ((A/a (0) A/a (t)), red), and donor-acceptor cross-correlation function ((A/d (0) A/d (t)), black), deduced from the single-molecule trajectories in (a). They are fitted with the same decay rate constant of 180 40s. A long decay component of 10 2s is also evident in each autocorrelation function. The first data point (not shown) of each correlation function contains the contribution from the measurement noise and fluctuations faster than the time resolution. The correlation functions are normalized, and the (A/a (0) A/a (t)) is presented with a shift on the y axis to enhance the view, (c) A pair of fluorescence trajectories from a donor (blue) and acceptor (red) pair in a T4 lysozyme protein without substrates present. The acceptor was photo-bleached at about 8.5 s. (d) The correlation functions (C(t)) of donor ((A/d (0) A/d (t)), blue), acceptor ((A/a (0) A/a (t)), red) derived from the trajectories in (c). The autocorrelation function only shows a spike at t = 0 and drops to zero at t > 0, which indicates that only uncorrelated measurement noise and fluctuation faster than the time resolution recorded (Adapted with permission from [12]. Copyright 2003 American Chemical Society)...

Examples of fluorescence labels for hgands are carboxyfluorescein, Cy3, a commercially available fluorescent marker based on a cyanine dye or tetramethyl-rhodamine. They are chemically introduced into a ligand. As with the radioactive labels, a possible influence of the labels on the binding behavior of the labeled hgands has to be considered, especially as the fluorescent dyes are complex molecules. Furthermore, the receptors themselves can be fluorescent labeled, which is done recombinantly. The respective receptors are expressed as fusion proteins with fluorescent proteins, e.g., green fluorescent protein (GFP) from Aequorea victoria, one of its mutant variants, or DSRed from Discosoma striata [26, 35]. 

Figure 5 Fluorescence signal from cuvettes with constant concentration of gold nanopaiticles but altering concentration of tetramethyl-Rhodamine. Assuming that one Rhodamine molecule occupies an area of-1 nm one can calculate the surface coverage for each concentration. Below 100 % surhice coverage the fluorescence is quenched, while above the fluorescence intensity increases linearly with Rhodamine concentration.

Four forms of amine-reactive rhodamine probes are commonly available. Two of them are based on the tetramethyl derivatives of the fundamental rhodamine structure, one is based on the sulforhodamine B or Lissamine derivative, and the last is the sulforhodamine 101 or Texas Red-type of derivative. All of them react under alkaline conditions with primary amines in proteins and other molecules to form stable, highly fluorescent complexes. 

In contrast, much more studies have been devoted to the ditfusion of small molecules in the multilayers, because of its importance in applications like permeation membranes or biosensing. Both IR spectroscopy [316] and fluorescence measurements [317] have shown the diffusion of protons in the multilayers, and thus an influence of the pH of the outer solution even far inside the films. The influence of water on the thickness of the multilayers is also well-documented [111,318]. Diffusion of radiolabeled salt ions has also been measured [125,312], Voltamperometry showed that PAH/PAA films had little effect on the diffusion of Fe(CN)g , but that PAH/PSS films could hinder its transport [116], 6-CF [96], acridine orange [81], daunomycin [251], 2 -3 cyclic adenosine monophosphate [252], bisulfite [313], and different diazonium salts [147,313] have been shown to permeate deeply in multilayers built by ESA. Immunoglobulin G (IgG) could permeate or not in a superlattice made of anti-IgG layers and PAH/PSS spacer layers, depending on the thickness of the spacer layer. The diffusion constants of rhodamine and of 2,2,6,6,-tetramethyl-4-piperidinol-l-oxide (TEMPOL) in PAH/PSS multilayers have been quantified [113,114],... 

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