Fluorescence polarization — a new tool for cell and molecular biology

Checovich, W J; Bolger, Randall E.; Burke, Thomas G.

doi:10.1038/375254a0

Cited by 175 publications

(114 citation statements)

References 8 publications

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“…Both assays incorporated fluorescence polarization technology to quantify the difference in anisotropy of a fluorescent tracer between its free and protein-bound states [30], [31] and [32]. The enzyme assay exploits the observation that DTB, enzymatically produced by DTBS, effectively competes with commercially available fluorescein-biotin (M r 831 Da) to bind streptavidin (M r 67 000 Da).…”

Section: Enzyme Activity and Assay Developmentmentioning

confidence: 99%

Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase

et al. 2015

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In all cases accepted manuscripts should:  link to the formal publication via its DOI  bear a CC-BY-NC-ND license -this is easy to do, click here to find out how  if aggregated with other manuscripts, for example in a repository or other site, be shared in alignment with our hosting policy  not be added to or enhanced in any way to appear more like, or to substitute for, the published journal article Embargo 1472-9792Tuberculosis 12months Here we provide evidence to show that MtDTBS has a broad nucleotide specificity due to the absence of the gate-keeper residue.

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Section: Enzyme Activity and Assay Developmentmentioning

confidence: 99%

Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase

et al. 2015

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“…The reaction mixture was poured into a 1:1 mixture of Et 2 O:EtOAc (150 mL), washed with 1 M HCl (2 x 100 mL) and brine (100 mL). The organic layer was dried over MgSO 4 and concentrated in vacuo. The crude oil was purified by flash chromatography on silica gel using a 10% to 40% gradient of EtOAc in hexane to recover 7.27 g of colorless oil which slowly crystallized over several days (92% yield).…”

Section: Synthetic Detailsmentioning

confidence: 99%

“…The organic phase was dried over MgSO 4 , concentrated in vacuo and purified by flash chromatography on silica gel, eluting with a gradient from 20% to 50% of EtOAc in hexane to recover 5.37 g (76% yield). This material (18.7 mmol) was dissolved in THF (50 mL), cooled to 0 °C and reacted with NaBH 4 (1.09 g, 18.7 mmol, 1.0 equiv) followed by MeOH (4.7 mL, 187 mmol, 10 equiv).…”

Section: Synthetic Detailsmentioning

confidence: 99%

“…Compound 11 (1.1 g, 1.12 mmol, 1.0 equiv) was dissolved in CH 2 Cl 2 (5 mL) at 23 °C, and Pd(PPh 3 ) 4 (0.129 g, 0.112 mmol, 0.1 equiv) was added followed by tributyltin hydride (0.492 g, 1.68 mmol, 1.5 equiv). After 3 hrs, the reaction was concentrated onto silica and loaded directly onto a short column.…”

Section: Synthetic Detailsmentioning

confidence: 99%

“…[1][2][3] Various detection methodologies for high-throughput screening (HTS) applications, including laser-induced fluorescence, fluorescence polarization, and fluorescence energy transfer, have been implemented in high density microtiter plate format. [4][5][6][7] The spatial separation of possible interacting pairs distinguishes signals from different ligand-target combinations, and readings are made by scanning techniques or by imaging. 8 At present, particular challenges exist in the development of multiplexed assays, which allow multiple interactions in samples to be detected simultaneously.…”

Section: Introductionmentioning

confidence: 99%

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Development of a Functionalized Xenon Biosensor

et al. 2004

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NMR-based biosensors that utilize laser-polarized xenon offer potential advantages beyond current sensing technologies. These advantages include the capacity to simultaneously detect multiple analytes, the applicability to in vivo spectroscopy and imaging, and the possibility of "remote" amplified detection. Here we present a detailed NMR characterization of the binding of a biotin-derivatized caged-xenon sensor to avidin. Binding of "functionalized" xenon to avidin leads to a change in the chemical shift of the encapsulated xenon in addition to a broadening of the resonance, both of which serve as NMR markers of ligand-target interaction. A control experiment in which the biotin-binding site of avidin was blocked with native biotin showed no such spectral changes, confirming that only specific binding, rather than nonspecific contact, between avidin and functionalized xenon leads to the effects on the xenon NMR spectrum. The exchange rate of xenon (between solution and cage) and the xenon spin-lattice relaxation rate were not changed significantly upon binding. We describe two methods for enhancing the signal from functionalized xenon by exploiting the laser-polarized xenon magnetization reservoir. We also show that the xenon chemical shifts are distinct for xenon encapsulated in different diastereomeric cage molecules. This demonstrates the potential for tuning the encapsulated xenon chemical shift, which is a key requirement for being able to multiplex the biosensor.

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High‐Throughput Screening

Turner

Sterrer

Wiesmüller

2000

Ullmann's Encyclopedia of Industrial Chemistry

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The article contains sections titled: 1. Introduction to Drug Discovery and Screening 1.1. A Rationale for new Methods in Drug Discovery 1.2. Sources of Compounds for High‐Throughput Screening 1.3. Demands on High‐Throughput Screening 2. Compounds 2.1. Novel Approaches to Generate Chemical Diversity 2.2. Microreactor Systems 2.3. Limitations of Pooled Compound Screening Strategies 3. Screening Technology 3.1. Chemistry, Biology, and Technology 3.2. Primary, Secondary, and Tertiary Screening 3.3. Miniaturization, Automation and Homogeneous Assays 3.4. Detection Technologies for High‐Throughput Screening 3.5. Examples of Specific Fluorescence Detection Technologies for High‐Throughput Screening 3.6. Case Study EVOscreen 4. Acknowledgements

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Fluorescence polarization — a new tool for cell and molecular biology

Abstract: Fluorescence polarization (FP) equilibrium binding assays differ from other types of binding studies in one important regard: they require no steps to separate free from bound tracer and are therefore fast, simple and accurate.

Cited by 175 publications

References 8 publications

Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase

Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase

Development of a Functionalized Xenon Biosensor

High‐Throughput Screening

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