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Single Molecule Studies Of Enzymes Horseradish Peroxidase And Alkaline Phosphatase Using Total Internal Reflection Fluorescence Microscopy And Confocal Microscopy
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Book Synopsis SINGLE MOLECULE STUDIES OF ENZYMES HORSERADISH PEROXIDASE AND ALKALINE PHOSPHATASE USING TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY AND CONFOCAL MICROSCOPY by : Leonora Kaldaras
Download or read book SINGLE MOLECULE STUDIES OF ENZYMES HORSERADISH PEROXIDASE AND ALKALINE PHOSPHATASE USING TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY AND CONFOCAL MICROSCOPY written by Leonora Kaldaras and published by . This book was released on 2013 with total page 46 pages. Available in PDF, EPUB and Kindle. Book excerpt: Conformational dynamics plays an important role in determining protein reactivity including affinity to substrate and product, catalytic rate constant and product release process. Whereas the influence of conformational dynamics on kinetic parameters of enzymatic reaction have been widely studied by ensemble averaging techniques, in these experiments conformational variations tend to average out and therefore ensemble averaging methods lack the level of resolution necessary to characterize time dependent behavior of individual enzyme molecules, which is essential to understanding enzymatic catalysis. This thesis consists of two parts. The first part focuses on the new development of integrated spectroscopy system, which combined the advantages of wide plane view field of objective-type TIRFM imaging with high time-resolution confocal single-molecule spectroscopy to address the challenge of probing both temporally and spatially stochastic events of the single-molecule reactions, such as fluorogenic enzymatic reactions with enzyme tethered to a solution/glass interface. Tthe calibration of this integrated apparatus is demonstrated with fluorescence microspheres and the feasibility by spectroscopy measurement of single molecule Rhodamine 6G on glass surface. Finally, the application of the apparatus is demonstrated by studying spatially and temporally randomly distributed single-molecule enzymatic reaction of fluorogenic substrate, horseradish peroxidase-catalyzed oxidation of amplex red. The second part focuses on exploratory studies of enzyme Alkaline Phosphatase from E. coli and its potential to be studied on single molecule level using fluorogenic substrate 3-o-methylfluorescein phosphate (MFP) and two polarization total internal reflection fluorescence microscopy apparatus. Ensemble averaged kinetics data and several single molecule trajectories are presented here.
Book Synopsis Investigation of Some Technical Aspects of Horseradish Peroxidase and Alkaline Phosphatase Conjugates for Use in ELISA Tests [microform] by : Robin Kim King
Download or read book Investigation of Some Technical Aspects of Horseradish Peroxidase and Alkaline Phosphatase Conjugates for Use in ELISA Tests [microform] written by Robin Kim King and published by National Library of Canada. This book was released on 1982 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The following study was undertaken to produce enzyme-conjugates to be used for antigen and antibody detection of Coxsackieviruses B1-B6 and for the detection of Western Equine Encephalomyelitis virus in animal and arthropod tissues. Anti-Coxsackieviruses B1-B6 immune sera were obtained from guinea pigs immunized with viral antigens prepared in HeLa cells and anti-Western Equine Encephalomyelitis virus was prepared in mice using antigen prepared in suckling mouse brains. Antiguinea pig and anti-mouse enzyme-labelled conjugates were prepared using horseradish peroxidase and alkaline phosphatase by a periodate and a one-step glutaraldehyde conjugation procedure respectively. The horseradish peroxidase conjugate prepared failed to react in the ELISA test and yields were very low with little enzymatic activity. Conjugates prepared using alkaline phosphatase had enzymatic activities ranging from 195-510 IU/mL and were titred using polysterene microtitre plates coated with 10 yg/mL immunoglobulin. The conjugate titres correspondingly ranged from 1:100 to 1:500 depending on the enzymatic activity. Retention of the original enzymatic activity ranged from 16-65%. Passage of the conjugates through Sephacryl S-300 superfine gel filtration helped remove unconjugated enzyme and immunoglobulin from the conjugate. The alkaline phosphatase conjugates were used to titrate Coxsackieviruses B1-B6 using checkerboard titration systems. A positive test value was taken as two times the absorbance of the normal HeLa cell control provided the latter value was equal to or less than 0.150 units. The optimal antigen concentration was at a 10" 3 dilution of the infected HeLa cell cultures except for Coxsackievirus B3 which reacted optimally at a 10" 2 dilution. No reaction was evident in any of the control wells and their absorbances were zero. The antisera titres ranged from 1:80 to 1:1280 and these endpoints were clearly defined. Secondary experiments employing Coxsackie B-virus antigens in AGMK cell culture resulted in gross non-specific findings which were attributed to non-specific protein binding of the polysterene plates. Preliminary testing of the plates using 1% bovine albumin in the diluents employed during the assay failed to reduce the non-specificity of the plastics arid work is ongoing to solve the problem. The ELISA has shown applicability to Coxsackie B-virus detection and, likewise, should be an efficient method of detecting Western Equine Encephalomyelitis virus once the parameters of testing have been completely worked out.
Book Synopsis Horseradish Peroxidase by : Maarten Uzun
Download or read book Horseradish Peroxidase written by Maarten Uzun and published by . This book was released on 2019 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Immunochemical Studies on Horseradish Peroxidase by : James M. Conroy
Download or read book Immunochemical Studies on Horseradish Peroxidase written by James M. Conroy and published by . This book was released on 1975 with total page 302 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Confocal Microscopy by : Stephen W. Paddock
Download or read book Confocal Microscopy written by Stephen W. Paddock and published by Springer Science & Business Media. This book was released on 2008-02-03 with total page 446 pages. Available in PDF, EPUB and Kindle. Book excerpt: In Confocal Microscopy Methods and Protocols, Stephen Paddock and a highly skilled panel of experts lead the researcher using confocal techniques from the bench top, through the imaging process, to the journal page. They concisely describe all the key stages of confocal imaging-from tissue sampling methods, through the staining process, to the manipulation, presentation, and publication of the realized image. Written in a user-friendly, nontechnical style, the methods specifically cover most of the commonly used model organisms: worms, sea urchins, flies, plants, yeast, frogs, and zebrafish. Centered in the many biological applications of the confocal microscope, the book makes possible the successful imaging of both fixed and living specimens using primarily the laser scanning confocal microscope. The powerful hands-on methods collected in Confocal Microscopy Methods and Protocols will help even the novice to produce first-class cover-quality confocal images.
Download or read book Heme Peroxidases written by Emma Raven and published by Royal Society of Chemistry. This book was released on 2015-10-27 with total page 388 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book will provide an up to date handbook that is unique in its combination of both the general aspects of heme protein structure and the function in a single volume.
Book Synopsis Mechanistic Studies on Horseradish Peroxidase and Its Role in the Catalysis of Oxidation Reactions by : Raheleh Ravanfar
Download or read book Mechanistic Studies on Horseradish Peroxidase and Its Role in the Catalysis of Oxidation Reactions written by Raheleh Ravanfar and published by . This book was released on 2019 with total page 204 pages. Available in PDF, EPUB and Kindle. Book excerpt: The catalyzed oxidation reactions by horseradish peroxidase (HRP) comprise a conversion of the ferric state of the heme to the ferryl heme by hydrogen peroxide (H2O2). However, studies barely investigate the effect of other components in the reaction mixtures on HRP function. Thus, we desire to investigate the HRP-catalyzed oxidation reactions in exposure to histidine (His). Herein, we report the first use of His and HRP to initiate the photocatalytic oxidation reactions without the exogenous addition of hydrogen peroxide. We demonstrate that the oxidation reactions of organic molecules proceed 2-times faster using HRP/His system in comparison with HRP/H2O2 system. During this work, we noticed that non-aromatic amino acids are fluorescent in solid state. We also demonstrate that the polymorph A crystal of L-His contains hydrophobic domains within the structure's interior, which can serve as vehicles for the highly efficient entrapment and transport of hydrophobic small molecules. Despite the importance of H2O2 in the HRP-catalyzed reactions, the detection and quantification of low concentrations of H2O2 is still limited using current methods. In this regard, we demonstrate that high-valent iron-oxo intermediates of HRP are well suited to detect ultratrace amount of H2O2 impurities in alcohols in the range of 0.001-1000 $M using just a UV/Vis spectrophotometer. We monitor the optical spectra of low concentrations of HRP (0.1-1 $M) for the red shift in the Soret and Q-band regions upon the addition of alcohols, as well as the reversibility of this shift to the original wavelength over time due to the spontaneous decay of ferryl intermediates to the ferric state. The motivation for this thesis lie in an industrially-funded project on Cheddar cheese. The aim of this project was to obtain white whey from the yellow whey proteins recovered from the Cheddar cheese making process without using oxidative agents. To address this issue, we conducted several experiments which resulted in all fascinating chapters of this thesis based on our observations, while none of them solved the problem. Thus, we designed microcapsules, which release the cargo only when exposed to lipase during the ripening step, selectively coloring the cheese matrix and obtaining the white whey.
Book Synopsis Attempts to Immobilize Horseradish Peroxidase on Solid Phases. II. Studies of Enzyme-antibody Conjugates Linked Through Their Oligosaccharides Via Spacer Arms by : Rong-Jen Hwang
Download or read book Attempts to Immobilize Horseradish Peroxidase on Solid Phases. II. Studies of Enzyme-antibody Conjugates Linked Through Their Oligosaccharides Via Spacer Arms written by Rong-Jen Hwang and published by . This book was released on 1994 with total page 388 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Single-Molecule Enzymology: Nanomechanical Manipulation and Hybrid Methods by :
Download or read book Single-Molecule Enzymology: Nanomechanical Manipulation and Hybrid Methods written by and published by Academic Press. This book was released on 2017-01-03 with total page 486 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-Molecule Enzymology, Part B, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in single-molecule enzymology, and includes sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, and nanopore and tethered particle motion. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers research methods in single-molecule enzymology Contains sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, and nanopore and tethered particle motion
Book Synopsis Kinetic and Structural Consequences of Modifications to the Substrate Interaction Site of Horseradish Peroxidase C by : Gareth Allen Williams
Download or read book Kinetic and Structural Consequences of Modifications to the Substrate Interaction Site of Horseradish Peroxidase C written by Gareth Allen Williams and published by . This book was released on 2005 with total page 294 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Effect of Nanopulses on Horseradish Peroxidase, an Enzyme Involved in Electron Transfer Reactions by : Anil Kumar Kalluri
Download or read book Effect of Nanopulses on Horseradish Peroxidase, an Enzyme Involved in Electron Transfer Reactions written by Anil Kumar Kalluri and published by . This book was released on 2004 with total page 106 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Guide to Research Techniques in Neuroscience by : Matt Carter
Download or read book Guide to Research Techniques in Neuroscience written by Matt Carter and published by Academic Press. This book was released on 2022-03-26 with total page 416 pages. Available in PDF, EPUB and Kindle. Book excerpt: Modern neuroscience research is inherently multidisciplinary, with a wide variety of cutting edge new techniques to explore multiple levels of investigation. This Third Edition of Guide to Research Techniques in Neuroscience provides a comprehensive overview of classical and cutting edge methods including their utility, limitations, and how data are presented in the literature. This book can be used as an introduction to neuroscience techniques for anyone new to the field or as a reference for any neuroscientist while reading papers or attending talks. Nearly 200 updated full-color illustrations to clearly convey the theory and practice of neuroscience methods Expands on techniques from previous editions and covers many new techniques including in vivo calcium imaging, fiber photometry, RNA-Seq, brain spheroids, CRISPR-Cas9 genome editing, and more Clear, straightforward explanations of each technique for anyone new to the field A broad scope of methods, from noninvasive brain imaging in human subjects, to electrophysiology in animal models, to recombinant DNA technology in test tubes, to transfection of neurons in cell culture Detailed recommendations on where to find protocols and other resources for specific techniques "Walk-through" boxes that guide readers through experiments step-by-step
Book Synopsis Modern Methods in Analytical Morphology by : J. Gu
Download or read book Modern Methods in Analytical Morphology written by J. Gu and published by Springer Science & Business Media. This book was released on 2012-12-06 with total page 441 pages. Available in PDF, EPUB and Kindle. Book excerpt: While advances in modem medicine largely parallel our understanding of morphology, discoveries in morphology are propelled by developments of new tools and means to visualize and measure tissue elements. The invention of dissecting, light, fluorescence and electron microscopes together with advances in labeling and staining techniques are among the stepping stones of morphological progress. Today, we are in an exciting new era when classical morphology is being combined with developments from other disciplines. The combination of morphology and immunology resulted in immunocytochemistry; morphology and molecular biology led to in situ hybridization and in situ PCR. Adding computer science to morphology gave birth to image analysis. Combining laser technology and the microsope evolved into confocal microscope. For more than a decade, modem morphology has continued to develop by merging with other disciplines at a rate that is still gathering momentum, providing exciting and dynamic new frontiers for other biological fields. "Modem Methods in Analytical Morphology," based largely on the "First International Workshop on Modem Methods in Analytical Histochemistry, "is an updated review of the current trends in the field. It covers an extensive array of new technical developments in major disciplines of modem morphology. The authors are not only leaders in their fields but also have extensive "hands on" experience with "bench work. " Their chapters are written in a comprehensive manner including discussion of both theoretical considerations and practical applications to give the readers a broad view of the topics covered.
Book Synopsis Principles and Techniques of Biochemistry and Molecular Biology by : Keith Wilson
Download or read book Principles and Techniques of Biochemistry and Molecular Biology written by Keith Wilson and published by Cambridge University Press. This book was released on 2010-03-04 with total page 769 pages. Available in PDF, EPUB and Kindle. Book excerpt: Uniquely integrates the theory and practice of key experimental techniques for bioscience undergraduates. Now includes drug discovery and clinical biochemistry.
Book Synopsis Asymptotic Methods in Quantum Mechanics by : S.H. Patil
Download or read book Asymptotic Methods in Quantum Mechanics written by S.H. Patil and published by Springer Science & Business Media. This book was released on 2000-04-26 with total page 194 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book describes some general properties of wave functions, with an emphasis on their asymptotic behaviour. The asymptotic region is particularly important since it is the wave function in the outer region of an atom, a molecule or a nucleus, which is sensitive to external interaction. An analysis of these properties helps in constructing simple and compact wave functions and in developing a broad understanding of different aspects of the quantum mechanics of many-particle systems. As applications, wave functions with correct asymptotic forms are used to generate a large data base for susceptibilities, polarizabilities, interatomic potentials, and nuclear densities.
Book Synopsis Detection of Non-Amplified Genomic DNA by : Giuseppe Spoto
Download or read book Detection of Non-Amplified Genomic DNA written by Giuseppe Spoto and published by Springer Science & Business Media. This book was released on 2012-07-06 with total page 320 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book offers an overview of state-of-the-art in non amplified DNA detection methods and provides chemists, biochemists, biotechnologists and material scientists with an introduction to these methods. In fact all these fields have dedicated resources to the problem of nucleic acid detection, each contributing with their own specific methods and concepts. This book will explain the basic principles of the different non amplified DNA detection methods available, highlighting their respective advantages and limitations. Non-amplified DNA detection can be achieved by adopting different techniques. Such techniques have allowed the commercialization of innovative platforms for DNA detection that are expected to break into the DNA diagnostics market. The enhanced sensitivity required for the detection of non amplified genomic DNA has prompted new strategies that can achieve ultrasensitivity by combining specific materials with specific detection tools. Advanced materials play multiple roles in ultrasensitive detection. Optical and electrochemical detection tools are among the most widely investigated to analyze non amplified nucleic acids. Biosensors based on piezoelectric crystal have been also used to detect unamplified genomic DNA. The main scientific topics related to DNA diagnostics are discussed by an outstanding set of authors with proven experience in this field.
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