Fluorescence assays

Udenfried, S. Fluorescence Assay in Biology and Medicine. Academic Press, London-New York 1962. 

Another major second messenger in cells is calcium ion. Virtually any mammalian cell line can be used to measure transient calcium currents in fluorescence assays when cells are preloaded with an indicator dye that allows monitoring of changes in cytosolic calcium concentration. These responses can be observed in real time, but a characteristic of these responses is that they are transient. This may lead to problems with hemi-equilibria in antagonist studies whereby the maximal responses to agonists may be depressed in the presence of antagonists. These effects are discussed more fully in Chapter 6. 

Table I describes several of the fluorescent assays that have been used in our lab to study neutrophil activation. Fluorescein-labeled W-formylhexapeptide (FLPEP) has been used to characterize the ki- netics of ligand binding, dissociation, and internalization at 37°C (7,8). FLPEP is added to a suspension of cells, then receptor-bound and free FLPEP in solution are distinguished by adding antibody to fluorescein. This is a high-affinity antibody which binds free FLPEP within 1 s hut does not bind cell-bound FLPEP. When it binds the FLPEP, it quenches the fluorescein fluorescence. Hence the residual fluorescence after antibody addition represents FLPEP that is bound to the cell. Nonspecific binding is determined in cell suspensions that contain an excess of nonfluorescent peptide.

It should be pointed out that when using ethidium bromide the sensitivity of the assays varies depending on the physical state of the nucleic acids (see Table I). Ethidium does not discriminate between RNA and DNA, although dyes are available which bind DNA exclusively, so the relative amounts of each may be determined by taking two sets of measurements. Alternatively, nucleases (DNA-ase or RNA-ase) can be used to exclusively remove one or the other in a mixture. Nucleic acids from different sources (see Table II) also show a variation in sensitivity, and the fluorescence assay lacks the selectivity of the hybridization technique. Nevertheless, for rapid screening or quality-control applications the fluorescence assay is still the method of choice. 

In current practice the fluorescence assay is often followed by the use of hybridization techniques when more selectivity is required. We have for instance used the fluorescence techniques to obtain data on the nucleic acid content of malaria vaccine proteins produced in Escherichia coli. The rapid turnaround time of the fluorescence assay is particularly useful during the early stages of purification to determine the optimal process conditions. After the final process has been arrived at and a variety of methods used to assess the nucleic acid content (including the hybridization techniques), the fluorescence method can be developed for routine quality-control purposes. In certain cases, particularly at high protein concentrations, the dye may bind to the protein with... 

Analyses for the Saxitoxins. Early methods for analysis of the saxitoxins evolved from those used for toxin isolation and purification. The principal landmarks in the development of preparative separation techniques for the saxitoxins were 1) the employment of carboxylate cation exchange resins by Schantz et al. (82) 2) the use of the polyacrylamide gel Bio-Gel P2 by Buckley and by Shimizu (5,78) and 3) the development by Buckley of an effective TLC system, including a new solvent mixture and a new visualization technique (83). The solvent mixture, designated by Buckley as "E", remains the best for general resolution of the saxitoxins. The visualization method, oxidation of the saxitoxins on silica gel TLC plates to fluorescent degradation products with hydrogen peroxide and heat, is an adaptation of the Bates and Rapoport fluorescence assay for saxitoxin in solution. Curiously, while peroxide oxidation in solution provides little or no response for the N-l-hydroxy saxitoxins, peroxide spray on TLC plates is a sensitive test for all saxitoxin derivatives with the C-12 gemdiol intact. 

Mergny JL et al. (2001) Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay. Proc Natl Acad Sci USA 98(6) 3062-3067... 

Tatarets AL, Fedyunyayeva IA, Dyubko TS, Povrozin YA, Doroshenko AO, Terpetschnig EA, Patsenker LD (2006) Ring-substituted squaraine dyes as probes and labels for fluorescence assays. Anal Chim Acta 570 214-223... 

Kroes-Nijboer A, Lubbersen YS, Venema P, van der Linden E (2009) Thioflavin T fluorescence assay for [beta]-lactoglobulin fibrils hindered by DAPH. J Struct Biol 165(3) 140... 

Cencic, R., Yan, Y., and Pelletier, J. (2007). Homogenous time resolved fluorescence assay to identify modulators of cap-dependent translation initiation. Comb. Chem. High Throughput Screen 10, 181-188. 

The model results were compared with the HOx concentrations measured by the FAGE (Fluorescence Assay by Gas Expansion) technique during four days of clean Southern Ocean marine boundary layer (MBL) air. The models overestimated OH concentrations by about 10% on two days and about 20% on the other two days. HO2 concentrations were measured during two of these days and the models overestimated the measured concentrations by about 40%. Better agreement with measured HO2 was observed by using data from several MBL aerosol measurements to estimate the aerosol surface area and by increasing the HO2 uptake coefficient to unity. This reduced the modelled HO2 overestimate by 40%, with little effect on OH, because of the poor HO2 to OH conversion at the low ambient NOx concentrations. 

Hennig A, Florea M, Roth D, Enderle T, Nau WM (2007) Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore. Anal Biochem 360 255-265... 

Neelakandan PP, Hariharan M, Ramaiah D (2006) A supramolecular ON-OFF-ON fluorescence assay for selective recognition of GTP. J Am Chem Soc 128 11334-11335... 

Sun H, Feng F, Yu M, Wang S (2007) Analyte-induced aggregation of a water-soluble conjugated polymer for fluorescent assay of oxalic acid. Macromol Rapid Commun 28 1905-1911... 

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