Readout fluorescence

Fluorescence was measured with a Turner model 111 filter fluorometer. The excitation filter was a Corning 7-60 (365 nm primary wavelength). The emission filters were Wratten 65-A (495 nm primary wavelength) and 2-A (sharp-cutoff below 415 nm). A digital multimeter was connected to the recorder terminals of the fluorometer to provide digital readout. Fluorescence-quenching (FQ) titrations were performed in batches. Preliminary experiments indicated that quenching was independent of time (at least 26 hours) after 30 minutes. Equilibration times of 60 minutes were used. 

The truth most likely lies somewhere in between. Bender [67] published the most quantitative study to date on the success of HTS at Novartis. Several conclusions could be drawn. Particular target types and assay technologies have a great impact on screening success, and this was not always correlated to the number of identifying hits in the HTS runs. For assay formats used a minimum of five times, LC/MS readouts succeed 83% of the time, followed by FP assays, which succeed in 72% of the cases. TR-FRET showed a success rate of 70%, with FLIPR assays (61%), fluorescence intensity readouts (59%), and AlphaScreen (60%) performing... 

The processes of both seed formation and fibril extension are dependent on temperature and on peptide concentration, with 37°C being required for establishing equilibrium within 24 h with 30 pM Pi 4o- A full description of the assay system may be found elsewhere [97,117], A 4 h reaction time is typically within the linear portion of the time course. This nucleus-dependent assay detects mainly inhibitors that are substoichiometric with the monomeric peptide, which is present at high concentration. It is relatively insensitive to inhibitors that target the monomeric peptide. Whether the inhibitors interact with the growing end of a seed or with a low abundance conformational form of the p peptide that is competent to add to the seed is difficult to determine at this time. Similar dose-response curves are obtained for Congo Red as an inhibitor with either thioflavin T (ThT) fluorescence or filtration of radioiodinated peptide readouts (Fig. 4) Caveats in the interpretation of both the ThT and radiometric filtration assays for the evaluation of putative inhibitors are discussed elsewhere [97]. 

Fluorescence correlation spectroscopy (FCS) measures rates of diffusion, chemical reaction, and other dynamic processes of fluorescent molecules. These rates are deduced from measurements of fluorescence fluctuations that arise as molecules with specific fluorescence properties enter or leave an open sample volume by diffusion, by undergoing a chemical reaction, or by other transport or reaction processes. Studies of unfolded proteins benefit from the fact that FCS can provide information about rates of protein conformational change both by a direct readout from conformation-dependent fluorescence changes and by changes in diffusion coefficient. 

Figures 3 and 4 illustrate how G( r) changes with Keq in the fast reaction regime (tc -C to). For both figures, k and the equilibrium constant A e( = l

Recently, SETA BioMedicals has developed a new near-infrared squaraine-based label Seta-633, which can be used to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter [19]. This label exhibits lower quantum yields and shorter fluorescence lifetimes when free in solution, but these values substantially increase upon interaction with proteins, which is contrary to tracers like Cy5 or Alexa 647. It was demonstrated in a model assay that a biotinylated Seta-633 binds to anti-biotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 2.76 fold upon binding to a specific antibody (anti-biotin, MW =160 kDa), while the titration with BSA or nonspecific antibody does not result in a noticeable change in lifetime (Fig. 13). The label is compatible with readily available light sources (635 nm or 640 nm lasers) and filter sets (as for Cy5 or Alexa 647) and its... 

As for the relevant application, recently, a specific photochromic compound, 1,2-bis(2 -methyl-5 -phenyl-3 -thienyl)perfluorocyclopentene (BP-BTE), and the analogs of HBO, 2,5-bis(5,-0 rt-butyl-benzooxazol-2 -yl)hydroquinone (DHBO), were employed in the high-contrast, reversible, photochromic switching of fluorescence emission and its perfect nondestructive readout (Fig. 14). Due to the large... 

Fig. 14 The molecular structures of BP-BTE and DHBO above). Microsized erasable ESIPT-fluorescence photoimaging on a spin-coated BP-BTE/DHBO-loaded PMMA film and its nondestructive readout capability (a) initial open-form state (b) writing (c) erasing (d) rewriting and (e) continuous nondestructive reading under irradiation with relatively high-intensity 415 nm light (200 4W cm-2) for 30 min. The dark region represents the area irradiated with the 365 nm UV light below) (reprint from ref. [88], Copyright 2006 American Chemical Society)...

Lim SJ, Seo J, Park SY (2006) Photochromic switching of excited-state intramolecular proton-transfer (ESIPT) fluorescence a unique route to high-contrast memory switching and nondestructive readout. J Am Chem Soc 128 14542-14547... 

Expression systems with direct or indirect fluorescence readout... 

Naphthalene-2,3-dicarboxaldehyde Nicotinamide adenine dinucleotide N-Acetylneuraminic acid 4-Fluoro-7-nitrobenzoxadiazole Naphthalene-2,3-dicarboxaldehyde Nondestructive readout Near infrared Near infrared fluorescence Nuclear magnetic resonance 2-Nitrophenyl oxalate 1,1 -Oxalyldiimidazole Polycyclic aromatic hydrocarbon Principal component analysis Photosensitized chemiluminescence Pentachlorophenyl oxalate Polymerase chain reaction... 

Measurements of binding curves without influencing the equilibria can be performed if the readout for complex formation is correlated with a change in a macroscopic signal. This can be either a change in fluorescence intensity, fluorescence polarization, optical absorption, or heat of association (see next chapter). Assume an equilibrium... 

Many of the methods described above may suffer from the modifications of the proteins which are necessary to obtain a detectable readout for the interaction under investigation. As a matter of fact, fluorescent labels are attached in close vicinity to the site of interaction, as otherwise binding to a partner is not likely to be detectable. It is conceivable that such labels invoke altered binding properties. Similar skepticism and arguments hold for immobilizing a protein. Non-invasive labeling is possible with radioactive isotopes but these methods bear other disadvantages. 

One-third of these appeared in the actives for the assay, which was defined using a 30% inhibition threshold. The overall hit rate for the assay was 2.0% in other words, 20% of the actives were false positives due to extreme optical interference with the assay readout. This number rises depending on the level of initial fluorescence used as a cutoff in the selection scheme used by the project team at the time, almost half of the actives were rejected as false positives on the basis of the initial fluorescence readings. The corporate screening collection has been reformatted since then, and many of these problem compounds have been removed. 

Unlike fluorescence detection, MS-based detection methods maintain their sensitivity when moving from normal-bore chromatography columns to capillary and nano LC systems. MS-based bioassays are therefore particularly suited for miniaturization. Conventional assays are operated at reagent flow rates of 20-50 pL min. By using electrospray MS as readout, flow rates of 1 pL min and lower could be envisaged, which is particularly useful for assays comprising expensive reagents. 

Pope, A.J., Haupts, U.M., Moore, K.J. Homogenous fluorescence readouts for miniaturized high-throughput screening theory and practice. Drug Discov. Today 1999, 4, 350-362. 

Commonly, in vitro determination of HDAC activity is a manual assay utilizing a coupled two-step process, including enzymatic deacetylation of a substrate followed by reaction termination and readout [10]. Assays utilize nuclear extracts and substrates containing labeled (radioactive or fluorescent) acetylated histones. For the isotope-based assays, the enzymes are incubated with acetate-radiolabled histones prepared from chicken reticulocytes or chemically [ Hjacetylated peptide substrates, and the enzymatic activity is determined by liquid scintillation counting [11]. Alternatively, histones may be obtained from cells following treatment with [ H]acetyl-CoA [12]. The caveats of these approaches include the variability of prelabeled acetylated core histones within preparations, potential high costs, their labor-intensive nature and the presence of radioactive waste. 

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