Fluor secondary

Labeling with fluorescent phalloidins may be combined with immunostaining. In this case, the phalloidin-staining solution can be applied in a mixture with fluores-cently labeled secondary antibodies. Combination of immunostaining with fluorescent phalloidins and fluorescently counterstained nuclei are extremely useful in multiple labeling strategies to locate antigens of interest with specific components of the cell. 

Some substances, known as fluors or scintillants, respond to the ionizing effects of alpha and beta particles by emitting flashes of light (or scintillations). While they do not respond directly to gamma rays, they do respond to the secondary ionization effects that gamma rays produce and, as a result, provide a valuable detection system for all emissions. 

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. 

If the light emitted during the decay of F"j is still of a wavelength too short for efficient measurement by a PMT, a secondary fluor, F2, that accepts energy from F j may be added to the scintillation system. Equations 6.13 and 6.14 outline the continued energy transfer process and fluorescence of F2. 

The light, hv2, from F2 is of longer wavelength than hvx from F and is more efficiently detected by a PMT. Two widely used primary and secondary fluors are 2,5-diphenyloxazole (PPO) with an emission maximum of 380 nm and l,4-bis-2-(5-phenyloxazolyl)benzene (POPOP) with an emission maximum of 420 nm. 

Color quenching is a problem if chemical substances that absorb photons from the secondary fluors are present in the scintillation mixture. Since the secondary fluors emit light in the visible region between 410 and 420 nm, colored substances may absorb the emitted light before it is detected by the photocells. Radioactive samples may be treated to remove colored impurities before mixing with the scintillation solvent. 

A variety of unfunctionalized secondary alcohols, including saturated and unsaturated carbinols, are resolved by catalyst 25 with moderate to high selectivi-ties (fcrei=4 to >50, see Scheme 5) [25]. Octapeptide 25 was discovered by screening a split-pool library of peptide catalyst candidates for acylation of 1-phe-nylethanol (3), using a reactivity-based fluorescence screen [26], followed by structure optimization with directed libraries. While substrates with increased steric bulk about the alcohol are resolved with highest selectivities, even 2-butanol is resolved with modest selectivity (fcrei=4). Peptide-based catalysts have also been applied to the resolution of tertiary alcohols, a relatively unexplored area of nonenzymatic asymmetric acylation catalysis [27-29], By using a fluores-... 

Fluorogenic labelling of pesticides. The subject has been reviewed earlier by Lawrence and Frei (27). Labelling consists in replacing a proton or other atom of a pesticide with a so-called labelling compound such as dansyl chloride (V) or fluores-camine (VI). The former reacts with primary and secondary amines, phenols, some thiols and aliphatic alcohols. The latter reacts very selectively with primary amines. 

Add secondary Alexa Fluor 488 goat anti-mouse antibody (diluted in PBS, recommended dilution 1 200-1 1000) and incubate for 1 h at room temperature. 

Some scintillation cocktails may employ a secondary fluor that absorbs photons from the primary fluor decay. The excited secondary fluor then reemits new photons at a wavelength more favorable for detection of experimental materials in the scintillation cocktail or more favorable to the phototubes of the particular scintillation counter. However, most modern scintillation counters no longer require the use of a secondary fluor. 

Several approaches have been used to circumvent these problems. First, scintillation cocktails have been developed that will accept more water and associated hydrophilic compounds. Bray s solution (4 g PPO, 0.2 g POPOP, 60 g naphthalene, 20 ml ethylene glycol, 100 ml methanol, and dioxane up to 1 liter), Kinard solution (xylene, p-dioxane, ethanol (5 5 3) containing 0.5% PPO, 0.005% a-NPO, and 6% naphthalene), and ethanol systems (e.g., 3 parts ethanol, 4 parts 0.8% PPO, 0.01% POPOP in toluene) are notable examples. In these examples, PPO denotes 2,5-diphenyloxazole, POPOP denotes l,4-bis-2-(5-phenyloxazolyl)-benzene, and a-NPO denotes 2-(l-naphthyl)-5-phenyloxazole. (POPOP and a-NPO are secondary fluors and can usually be omitted with modern scintillation counters.)... 

Figure 3-5. Structures of widely used primary and secondary fluors.

The 3700A wavelength photons interact and excite the POPOP molecules. The POPOP in this case is called the secondary fluor or the -wavelength shifter, and... 

In tyramide signal amplification (TSA), the target cells are labelled with an antibody or a nucleic acid probe, followed by secondary detection with a horseradish peroxidase (HRP) labelled antibody. HRP activates multiple copies of fluores-cently labelled tyramide derivatives, yielding fluorescent tyramide radicals that are deposited in the vicinity of the HRP-target interaction site (Fig. 5). 

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