The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Vincent T Lee - One of the best experts on this subject based on the ideXlab platform.
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differential radial Capillary Action of ligand assay dracala
Current Protocols in Molecular Biology, 2019Co-Authors: Anna B Seminara, Asan Turdiev, Husan Turdiev, Vincent T LeeAbstract:Protein interActions with nucleic acids are important for the synthesis, regulation, and stability of macromolecules. While a number of assays are available for interrogating these interActions, the differential radial Capillary Action of ligand assay (DRaCALA) has been developed as an easy and flexible platform that allows for the study of individual interActions when carrying out high-throughput screening for novel binding proteins and small molecule inhibitors. In this article, we describe the principle of DRaCALA and methods that utilize DRaCALA to determine the affinity and specificity of individual protein-nucleic acid interActions as well as uses for screening for binding proteins and chemical inhibitors. © 2018 by John Wiley & Sons, Inc.
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differential radial Capillary Action of ligand assay dracala for high throughput detection of protein metabolite interActions in bacteria
Methods of Molecular Biology, 2017Co-Authors: Mona W Orr, Vincent T LeeAbstract:Bacteria rely on numerous nucleotide second messengers for signal transduction such as cyclic AMP, cyclic-di-GMP, and cyclic-di-AMP. Although a number of receptors responsible for known regulated phenotypes have been established, the completeness of protein receptors in any given organism remains elusive. We have developed a method called differential radial Capillary Action of ligand assay (DRaCALA) that allows for an unbiased, systematic high-throughput screen for the detection of ligand binding proteins encoded by a genome. DRaCALA permits interrogation of ligand binding directly to an overexpressed protein in a cell lysate and bypasses the need of protein purification. Gateway-cloning-compatible open reading frame libraries are available for a diverse range of bacterial species and permits generation of the lysates overexpressing each open reading frame. These lysates can be assessed by DRaCALA in a 96-well format to allow rapid identification of protein-ligand interActions, including previously unknown proteins. Here, we present the protocols for generating the expression library, conducting the DRaCALA screen, data analysis, and hit validation.
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differential radial Capillary Action of ligand assay for high throughput detection of protein metabolite interActions
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Kevin G Roelofs, Jingxin Wang, Herman O Sintim, Vincent T LeeAbstract:InterActions of proteins with low-molecular-weight ligands, such as metabolites, cofactors, and allosteric regulators, are important determinants of metabolism, gene regulation, and cellular homeostasis. Pharmaceuticals often target these interActions to interfere with regulatory pathways. We have developed a rapid, precise, and high-throughput method for quantitatively measuring protein-ligand interActions without the need to purify the protein when performed in cells with low background activity. This method, differential radial Capillary Action of ligand assay (DRaCALA), is based on the ability of dry nitrocellulose to separate the free ligand from bound protein–ligand complexes. Nitrocellulose sequesters proteins and bound ligand at the site of application, whereas free ligand is mobilized by bulk movement of the solvent through Capillary Action. We show here that DRaCALA allows detection of specific interActions between three nucleotides and their cognate binding proteins. DRaCALA allows quantitative measurement of the dissociation constant and the dissociation rate. Furthermore, DRaCALA can detect the expression of a cyclic-di-GMP (cdiGMP)-binding protein in whole-cell lysates of Escherichia coli, demonstrating the power of the method to bypass the prerequisite for protein purification. We have used DRaCALA to investigate cdiGMP signaling in 54 bacterial species from 37 genera and 7 eukaryotic species. These studies revealed the presence of potential cdiGMP-binding proteins in 21 species of bacteria, including 4 unsequenced species. The ease of obtaining metabolite-protein interAction data using the DRaCALA assay will facilitate rapid identification of protein-metabolite and protein-pharmaceutical interActions in a systematic and comprehensive approach.
Peter Myers - One of the best experts on this subject based on the ideXlab platform.
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Capillary Action liquid chromatography
Journal of Separation Science, 2009Co-Authors: Bo Zhang, Edmund T Bergstrom, David M Goodall, Peter MyersAbstract:Capillary Action LC (caLC) is introduced as a technique using Capillary Action as the driving force to perform LC in Capillary columns packed with HPLC type microparticulate materials. A dry packing method with centrifugal force was developed to prepare Capillary columns in parallel (10 columns per 3 min) to support their disposable use in caLC. Using a digital microscope for real-time imaging and recording separations of components in a dye mixture, caLC was found to have flow characteristics similar to TLC. Based on the investigation of microparticulate HPLC silica gels of different size (1.5–10 μm) and a typical TLC grade irregular medium, Merck 60G silica, the van Deemter curves suggested molecular diffusion as the major contribution to band broadening in caLC. With Waters Xbridge 2.6 μm silica, plate heights down to 8.8 μm were obtained, comparable to those achievable in HPLC. Assisted by an image-processing method, the visual caLC separation was converted to a classical chromatogram for further data analysis and such a facility confirmed the observation of highly efficient bands.
Yuchie Chen - One of the best experts on this subject based on the ideXlab platform.
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automatic sampling and analysis of organics and biomolecules by Capillary Action supported contactless atmospheric pressure ionization mass spectrometry
PLOS ONE, 2013Co-Authors: Cheng Huan Hsieh, Anil Kumar Meher, Yuchie ChenAbstract:Contactless atmospheric pressure ionization (C-API) method has been recently developed for mass spectrometric analysis. A tapered Capillary is used as both the sampling tube and spray emitter in C-API. No electric contact is required on the Capillary tip during C-API mass spectrometric analysis. The simple design of the ionization method enables the automation of the C-API sampling system. In this study, we propose an automatic C-API sampling system consisting of a Capillary (∼1 cm), an aluminium sample holder, and a movable XY stage for the mass spectrometric analysis of organics and biomolecules. The aluminium sample holder is controlled by the movable XY stage. The outlet of the C-API Capillary is placed in front of the orifice of a mass spectrometer, whereas the sample well on the sample holder is moved underneath the Capillary inlet. The sample droplet on the well can be readily infused into the C-API Capillary through Capillary Action. When the sample solution reaches the Capillary outlet, the sample spray is readily formed in the proximity of the mass spectrometer applied with a high electric field. The gas phase ions generated from the spray can be readily monitored by the mass spectrometer. We demonstrate that six samples can be analyzed in sequence within 3.5 min using this automatic C-API MS setup. Furthermore, the well containing the rinsing solvent is alternately arranged between the sample wells. Therefore, the C-API Capillary could be readily flushed between runs. No carryover problems are observed during the analyses. The sample volume required for the C-API MS analysis is minimal, with less than 1 nL of the sample solution being sufficient for analysis. The feasibility of using this setup for quantitative analysis is also demonstrated.
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Capillary Action supported contactless atmospheric pressure ionization for the combined sampling and mass spectrometric analysis of biomolecules
Analytical Chemistry, 2011Co-Authors: Cheng Huan Hsieh, Chia Hsien Chang, Pawel L Urban, Yuchie ChenAbstract:It is proposed that a short tapered Capillary can be utilized as a nanoliter-volume sampling tool and sample emitter for generation of gas-phase ions in front of the mass spectrometer, without the need for using an additional electric power supply, a gas supply, or a syringe pump. A wide range of molecules can be analyzed in pure solutions and complex matrixes (cell extract, urine, and plant tissue) with no or minimum sample preparation. Singly and multiply charged ions can be detected in either positive or negative-ion mode. Because of the nanoliter-volume sampling and low spectral background, the mass detection limit for bradykinin is in the low attomole range. Other advantages include simplicity, disposability, and low cost. The putative mechanism of the ion formation in this Capillary-Action supported contactless spray emitter is discussed.
Cheng Huan Hsieh - One of the best experts on this subject based on the ideXlab platform.
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automatic sampling and analysis of organics and biomolecules by Capillary Action supported contactless atmospheric pressure ionization mass spectrometry
PLOS ONE, 2013Co-Authors: Cheng Huan Hsieh, Anil Kumar Meher, Yuchie ChenAbstract:Contactless atmospheric pressure ionization (C-API) method has been recently developed for mass spectrometric analysis. A tapered Capillary is used as both the sampling tube and spray emitter in C-API. No electric contact is required on the Capillary tip during C-API mass spectrometric analysis. The simple design of the ionization method enables the automation of the C-API sampling system. In this study, we propose an automatic C-API sampling system consisting of a Capillary (∼1 cm), an aluminium sample holder, and a movable XY stage for the mass spectrometric analysis of organics and biomolecules. The aluminium sample holder is controlled by the movable XY stage. The outlet of the C-API Capillary is placed in front of the orifice of a mass spectrometer, whereas the sample well on the sample holder is moved underneath the Capillary inlet. The sample droplet on the well can be readily infused into the C-API Capillary through Capillary Action. When the sample solution reaches the Capillary outlet, the sample spray is readily formed in the proximity of the mass spectrometer applied with a high electric field. The gas phase ions generated from the spray can be readily monitored by the mass spectrometer. We demonstrate that six samples can be analyzed in sequence within 3.5 min using this automatic C-API MS setup. Furthermore, the well containing the rinsing solvent is alternately arranged between the sample wells. Therefore, the C-API Capillary could be readily flushed between runs. No carryover problems are observed during the analyses. The sample volume required for the C-API MS analysis is minimal, with less than 1 nL of the sample solution being sufficient for analysis. The feasibility of using this setup for quantitative analysis is also demonstrated.
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Capillary Action supported contactless atmospheric pressure ionization for the combined sampling and mass spectrometric analysis of biomolecules
Analytical Chemistry, 2011Co-Authors: Cheng Huan Hsieh, Chia Hsien Chang, Pawel L Urban, Yuchie ChenAbstract:It is proposed that a short tapered Capillary can be utilized as a nanoliter-volume sampling tool and sample emitter for generation of gas-phase ions in front of the mass spectrometer, without the need for using an additional electric power supply, a gas supply, or a syringe pump. A wide range of molecules can be analyzed in pure solutions and complex matrixes (cell extract, urine, and plant tissue) with no or minimum sample preparation. Singly and multiply charged ions can be detected in either positive or negative-ion mode. Because of the nanoliter-volume sampling and low spectral background, the mass detection limit for bradykinin is in the low attomole range. Other advantages include simplicity, disposability, and low cost. The putative mechanism of the ion formation in this Capillary-Action supported contactless spray emitter is discussed.
Kevin T Brumund - One of the best experts on this subject based on the ideXlab platform.
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needle biopsy of routine thyroid nodules should be performed using a Capillary Action technique with 24 to 27 gauge needles a systematic review and meta analysis
Thyroid, 2018Co-Authors: William J Moss, Andrey Finegersh, Charles S Coffey, John Pang, Joseph A Califano, Ryan K Orosco, Kevin T BrumundAbstract:Background: Fine-needle biopsy is the international standard for diagnostic evaluation of thyroid nodules. There is a lack of consensus regarding the optimal needle biopsy technique. The implementation of Capillary Action versus aspiration and the optimal needle gauge remain topics of debate. Methods: A systematic review of the Medline, Embase, and Cochrane databases was performed in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Articles evaluating the effects of Capillary Action versus aspiration and needle gauge on success rates of fine-needle biopsy of the thyroid were assessed for inclusion. The primary outcome of interest was the rate of non-diagnostic cytopathology. Results: Twenty-four articles with a collective 4428 nodules were ultimately included in the review. Twenty articles evaluated Capillary Action versus aspiration, and six evaluated needle gauge. All but two studies were prospective, most of which were blinded trials with or without randomi...
