Download or read book Development of New Fluorescent Protein Biosensors written by Ahmed Saied F. Belal and published by . This book was released on 2014 with total page 124 pages. Available in PDF, EPUB and Kindle. Book excerpt: The discovery of green fluorescent protein (GFP) from the Aequorea victoria Jellyfish followed by the extensive efforts of protein engineers to produce other fluorescent proteins (FPs) spanning the visible color spectrum made fluorescent proteins indispensable biochemical tools in the scientific community. Experimental biologists have utilized FPs as genetically encoded markers for the imaging of subcellular structures and protein dynamics in live cells. Another important application of FPs is their design for use in biosensors for either enzymes or small biological molecules of interest. The work described in this thesis is an attempt to portray different experimental designs of FP based biosensors with the final objective of either modifying previously reported or introducing novel biosensors. We addressed the FP biosensors based on the principle of intramolecular Förster resonance energy transfer (FRET) in two projects. In the first project we demonstrated a modification of a methodology of development and optimization of FRET-based biosensor for a post translational modification. The end result of this project has led to improving a previously reported protein kinase B (PkB) biosensor and the discovery of a new cyclin B1- cyclin dependent kinase 1 (Cyclin B1-CDK1) biosensor of a higher dynamic range than previously published one. In the second project our efforts were directed to develop a matrix metallo proteinase 2 (MMP2) FRET based biosensor, with the ultimate goal of using this biosensor in live cell imaging of cardiomyocytes to explore postulated MMP2 intracellular role in ischemia-reperfusion injury. We designed protein constructs based on both previously reported MMP2 substrate sequences and potential cardiac protein target sequences. After testing and characterizing the designed constructs, the expression of the best candidate in neonatal cardiomyocytes cell lines is undertaken by our collaborator Professor Richard Schulz, Department of Pharmacology, University of Alberta. In the third and final project we utilized single FP-based biosensor concept in trying to develop Hydrogen peroxide biosensors of different hues. Our efforts were fruitful in discovering two green biosensors, one is with direct and the other is with inverse response to hydrogen peroxide.
Download or read book Fluorescence Based Biosensors written by and published by Academic Press. This book was released on 2012-12-31 with total page 449 pages. Available in PDF, EPUB and Kindle. Book excerpt: One of the major challenges of modern biology and medicine consists in finding means to visualize biomolecules in their natural environment with the greatest level of accuracy, so as to gain insight into their properties and behaviour in a physiological and pathological setting. This has been achieved thanks to the design of novel imaging agents, in particular to fluorescent biosensors. Fluorescence Biosensors comprise a large set of tools which are useful for fundamental purposes as well as for applications in biomedicine, drug discovery and biotechnology. These tools have been designed and engineered thanks to the combined efforts of chemists and biologists over the last decade, and developed hand in hand together with imaging technologies. This volume will convey the many exciting developments the field of fluorescent biosensors and reporters has witnessed over the recent years, from concepts to applications, including chapters on the chemistry of fluorescent probes, on technologies for monitoring protein/protein interactions and technologies for imaging biosensors in cultured cells and in vivo. Other chapters are devoted to specific examples of genetically-encoded reporters, or to protein and peptide biosensors, together with examples illustrating their application to cellular and in vivo imaging, biomedical applications, drug discovery and high throughput screening. - Contributions from leading authorities - Informs and updates on all the latest developments in the field
Download or read book Green Fluorescent Protein written by Martin Chalfie and published by John Wiley & Sons. This book was released on 2005-11-18 with total page 486 pages. Available in PDF, EPUB and Kindle. Book excerpt: Since the discovery of the gene for green fluorescent protein (GFP), derived from jellyfish, this protein that emits a green glow has initiated a revolution in molecular biosciences. With this tool, it is now possible to visualize nearly any protein of interest in any cell or tissue of any species. Since the publication of the first edition, there have been tremendously significant technological advances, including development of new mutant variants. Proteins are now available in yellow and blue, and Novel Fluorescent Proteins (NFPs) have expanded their utility in developing biosensors, biological markers, and other biological applications. This updated, expanded new edition places emphasis on the rise of NFPs, including new chapters on NFP properties with detailed protocols, applications of GFPs and NFPs in industry research, and biosensors. This book provides a solid theoretical framework, along with detailed, practical guidance on use of GFPs and NFPs with discussion of potential pitfalls. The expert contributors provide real examples in showing how to tailor GFP/NFP to specific systems, maximize expression, and enhance detection.
Download or read book Fluorescent Protein Based Biosensors written by Jin Zhang and published by Humana. This book was released on 2013-09-20 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: In Fluorescent Protein-Based Biosensors: Methods and Protocols, experts in the field have assembled a series of protocols describing several methods in which fluorescent protein-based reporters can be used to gain unique insights into the regulation of cellular signal transduction. Genetically encodable fluorescent biosensors have allowed researchers to observe biochemical processes within the endogenous cellular environment with unprecedented spatiotemporal resolution. As the number and diversity of available biosensors grows, it is increasingly important to equip researchers with an understanding of the key concepts underlying the design and application of genetically encodable fluorescent biosensors to live cell imaging. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Fluorescent Protein-Based Biosensors: Methods and Protocols promises to be a valuable resource for researchers interested in applying current biosensors to the study of biochemical processes in living cells as well as those interested in developing novel biosensors to visualize other cellular phenomena.
Download or read book Fluorescent Protein Based Biosensors written by Jin Zhang and published by Humana. This book was released on 2016-08-23 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: In Fluorescent Protein-Based Biosensors: Methods and Protocols, experts in the field have assembled a series of protocols describing several methods in which fluorescent protein-based reporters can be used to gain unique insights into the regulation of cellular signal transduction. Genetically encodable fluorescent biosensors have allowed researchers to observe biochemical processes within the endogenous cellular environment with unprecedented spatiotemporal resolution. As the number and diversity of available biosensors grows, it is increasingly important to equip researchers with an understanding of the key concepts underlying the design and application of genetically encodable fluorescent biosensors to live cell imaging. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Fluorescent Protein-Based Biosensors: Methods and Protocols promises to be a valuable resource for researchers interested in applying current biosensors to the study of biochemical processes in living cells as well as those interested in developing novel biosensors to visualize other cellular phenomena.
Download or read book Metal Enhanced Fluorescence written by Chris D. Geddes and published by John Wiley & Sons. This book was released on 2010-06-22 with total page 655 pages. Available in PDF, EPUB and Kindle. Book excerpt: Discover how metal-enhanced fluorescence is changing traditional concepts of fluorescence This book collects and analyzes all the current trends, opinions, and emerging hot topics in the field of metal-enhanced fluorescence (MEF). Readers learn how this emerging technology enhances the utility of current fluorescence-based approaches. For example, MEF can be used to better detect and track specific molecules that may be present in very low quantities in either clinical samples or biological systems. Author Chris Geddes, a noted pioneer in the field, not only explains the fundamentals of metal-enhanced fluorescence, but also the significance of all the most recent findings and models in the field. Metal-enhanced fluorescence refers to the use of metal colloids and nanoscale metallic particles in fluorescence systems. It offers researchers the opportunity to modify the basic properties of fluorophores in both near- and far-field fluorescence formats. Benefits of metal-enhanced fluorescence compared to traditional fluorescence include: Increased efficiency of fluorescence emission Increased detection sensitivity Protect against fluorophore photobleaching Applicability to almost any molecule, including both intrinsic and extrinsic chromophores Following a discussion of the principles and fundamentals, the author examines the process and applications of metal-enhanced fluorescence. Throughout the book, references lead to the primary literature, facilitating in-depth investigations into particular topics. Guiding readers from the basics to state-of-the-technology applications, this book is recommended for all chemists, physicists, and biomedical engineers working in the field of fluorescence.
Download or read book Fluorescent Proteins written by Kevin F. Sullivan and published by Elsevier. This book was released on 2007-12-14 with total page 613 pages. Available in PDF, EPUB and Kindle. Book excerpt: This new edition of Fluorescent Proteins presents current applications of autofluorescent proteins in cell and molecular biology authored by researchers from many of the key laboratories in the field. Starting from a current review of the broad palette of fluorescent proteins available, several chapters focus on key autofluorescent protein variants, including spectral variants, photodynamic variants as well as chimeric FP approaches. Molecular applications are addressed in chapters that detail work with single molecules, approaches to generating protein fusions and biosensors as well as analysis of protein-protein interactions in vivo by FRET, fluorescence polarization and fluorescence cross correlation techniques. A number of approaches to in vivo dynamics are presented, including FRAP, photoactivation, and 4-dimensional microscopy. Behavior of spindle components, membrane proteins, mRNA trafficking as well as analysis of cell types in tissues and in development are detailed and provide models for a wide variety of experimental approaches. In addition, several chapters deal directly with the computational issues involved in processing multidimensional image data and using fluorescent imaging to probe cellular behavior with quantitative modeling. This volume brings together the latest perspective and techniques on fluorescent proteins and will be an invaluable reference in a wide range of laboratories.
Download or read book Protein Engineering written by Huimin Zhao and published by John Wiley & Sons. This book was released on 2021-08-23 with total page 41 pages. Available in PDF, EPUB and Kindle. Book excerpt: A one-stop reference that reviews protein design strategies to applications in industrial and medical biotechnology Protein Engineering: Tools and Applications is a comprehensive resource that offers a systematic and comprehensive review of the most recent advances in the field, and contains detailed information on the methodologies and strategies behind these approaches. The authors—noted experts on the topic—explore the distinctive advantages and disadvantages of the presented methodologies and strategies in a targeted and focused manner that allows for the adaptation and implementation of the strategies for new applications. The book contains information on the directed evolution, rational design, and semi-rational design of proteins and offers a review of the most recent applications in industrial and medical biotechnology. This important book: Covers technologies and methodologies used in protein engineering Includes the strategies behind the approaches, designed to help with the adaptation and implementation of these strategies for new applications Offers a comprehensive and thorough treatment of protein engineering from primary strategies to applications in industrial and medical biotechnology Presents cutting edge advances in the continuously evolving field of protein engineering Written for students and professionals of bioengineering, biotechnology, biochemistry, Protein Engineering: Tools and Applications offers an essential resource to the design strategies in protein engineering and reviews recent applications.
Download or read book Protein Based Fluorescent Biosensors for Small Molecule Analytes written by Nissa M. Westerberg and published by . This book was released on 2005 with total page 534 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Protein Engineering Design Principles for the Development of Biosensors written by Seymour De Picciotto and published by . This book was released on 2015 with total page 136 pages. Available in PDF, EPUB and Kindle. Book excerpt: Investigating protein location and concentration is critical to understanding function. Reagentless biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can detect analytes of interest with high temporal and spatial resolution. However, because these biosensors require laborious empirical screening to develop, their adoption has been limited. Hence, we establish design principles that will facilitate development. In this thesis, we first develop a kinetic model for the dynamic performance of a reagentless biosensor. Using a sinusoidal signal for ligand concentration, our findings suggest that it is optimal to use a binding moiety whose equilibrium dissociation constant matches that of the average predicted input signal, while maximizing both the association rate constant and the dissociation rate constant at the necessary ratio to create the desired equilibrium constant. Although practical limitations constrain the attainment of these objectives, the derivation of these design principles provides guidance for improved reagentless biosensor performance and metrics for quality standards in the development of biosensors. Following these guidelines, we use the human tenth type III fibronectin domain to engineer new binders against several ligands of the EGFR receptor. Using these binders and others, we design and characterize biosensors based on various target analytes, scaffolds and fluorophores. We observe that analytes can harbor specific binding pockets for the fluorophore, which sharply increase the fluorescence produced upon binding. Furthermore, we demonstrate that a fluorophore conjugated to locally rigid surfaces possesses lower background fluorescence. Based on these newly identified properties, we design biosensors that produce a 100-fold increase in fluorescence upon binding to analyte, about a 10-fold improvement over the previous best biosensor. In order to improve the methodology of reagentless biosensor design, we establish a method for site-specific labeling of proteins displayed on the surface of yeasts. This procedure allows for the screening of libraries of sensors for binding and fluorescence enhancement simultaneously. Finally, we explore an alternative sensor design, based on competitive inhibition of fluorescence quenching.
Download or read book Fluorescent Proteins II written by Gregor Jung and published by Springer Science & Business Media. This book was released on 2012-01-05 with total page 287 pages. Available in PDF, EPUB and Kindle. Book excerpt: Fluorescent proteins are intimately connected to research in the life sciences. Tagging of gene products with fluorescent proteins has revolutionized all areas of biosciences, ranging from fundamental biochemistry to clinical oncology, to environmental research. The discovery of the Green Fluorescent Protein, its first, seminal application and the ingenious development of a broad palette of fluorescence proteins of other colours, was consequently recognised with the Nobel Prize for Chemistry in 2008. Fluorescent Proteins II highlights the physicochemical and biophysical aspects of fluorescent protein technology beyond imaging. It is tailored to meet the needs of physicists, chemists and biologists who are interested in the fundamental properties of fluorescent proteins, while also focussing on specific applications. The implementations described are cutting-edge studies and exemplify how the physical and chemical properties of fluorescent proteins can stimulate novel findings in life sciences.
Download or read book The Fluorescent Protein Revolution written by Richard N. Day and published by CRC Press. This book was released on 2014-04-28 with total page 350 pages. Available in PDF, EPUB and Kindle. Book excerpt: Advances in fluorescent proteins, live-cell imaging, and superresolution instrumentation have ushered in a new era of investigations in cell biology, medicine, and physiology. From the identification of the green fluorescent protein in the jellyfish Aequorea victoria to the engineering of novel fluorescent proteins, The Fluorescent Protein Revolution explores the history, properties, and applications of these important probes. The book first traces the history of fluorescent proteins and the revolution they enabled in cellular imaging. It then discusses fluorescent proteins with novel photophysical properties. The book also covers several cutting-edge imaging applications. These include superresolution microscopy of cellular fine structures, FRET microscopy to visualize protein interactions and cell-signaling activities inside living cells, photobleaching and photoactivation techniques to visualize protein behaviors, techniques that exploit plant and algal photoreceptors to enable light-regulated control of enzymatic activities, and the noninvasive imaging of tumor–host interactions in living animals. In color throughout, this book presents the fundamental principles and latest advances in the field, including the associated development of imaging techniques that exploit fluorescent proteins. It is accessible to a broad audience, from optical imaging experts to novices needing an introduction to the field.
Download or read book Fluorescence Sensors and Biosensors written by Richard B. Thompson and published by CRC Press. This book was released on 2005-12-16 with total page 416 pages. Available in PDF, EPUB and Kindle. Book excerpt: Fluorescence-based sensing is a significant technique used in prominent fields such as fluorescence-activated cell sorting, DNA sequencing, high-throughput screening, and clinical diagnostics. Fluorescence Sensors and Biosensors emphasizes the most recent developments and emerging technologies with the broadest impacts. The text begins wi
Download or read book Expanding Spectral Range and Enhancing Throughput of Fluorescent Biosensor Imaging written by Clara Anne Posner and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cells execute specific responses to diverse environmental cues by encoding information in distinctly compartmentalized biochemical signaling reactions. Genetically encoded fluorescent biosensors enable the spatial and temporal monitoring of signaling events in live cells. Chapter 1 of this dissertation is a reprint of work submitted for publication, that introduces the concepts of compartmentalized signaling, genetically encoded fluorescent biosensors, and temporal and spatiotemporal deterministic computational modeling and then demonstrates the power of applying computational models to interpret biosensor experiments in complex biochemical networks in two case studies. Chapter 2 of this dissertation is a reprint of published work in which we demonstrate that a mutation on a solvent-exposed residue of a red fluorescent protein (RFP) leads to a brighter, more stably fluorescent RFP. We also show that this RFP enhances green-red FRET biosensors and can be incorporated into a spectrally orthogonal FRET biosensor multiplexing scheme to investigate the crosstalk between Src, Akt, and Extracellular Regulated Kinase (ERK) kinases in single cells. Chapter 3 is a reprint of published work wherein we demonstrate that a platform combing an open-source liquid handler with a fluorescent microscope can automate and enhance throughput of fluorescent biosensor imaging experiments in response to multiple compound stimulations. We demonstrate this platform using protein kinase A (PKA) and 3',5'-cyclic adenosine monophosphate (cAMP) FRET biosensors to measure the signaling dynamics in response to varying concentrations of a single G-protein coupled receptor (GPCR) agonist, to a panel of GPCR agonists and antagonists, or to pH media exchanges. Finally, Chapter 4 is a conclusion where we summarize the findings and discuss future extensions of the work. This work describes three different methods that increase the utility of fluorescent biosensors experiments to investigate cell signaling phenomenon: quantitatively evaluating biosensor experimental data using computational models, developing a new red fluorescent protein and a three FRET biosensor multiplexing scheme, and engineering a platform that automates and increases throughput of fluorescent biosensor imaging experiments.
Download or read book High Content Screening written by D. Lansing Taylor and published by Springer Science & Business Media. This book was released on 2008-02-04 with total page 437 pages. Available in PDF, EPUB and Kindle. Book excerpt: There has always been some tension between proponents of hypothesis-driven and discovery-driven research in the broad field of life sciences. Academic research has been primarily focused on hypothesis-driven research. However, the success of the human genome project, a discovery-driven research approach, has opened the door to adding other types of discovery-driven research to a continuum of research approaches. In contrast, drug discovery research in the pharmaceutical industry has embraced discovery-driven research for many years. A good example has been the discovery of active compounds from large chemical libraries, through screening campaigns. The success of the human genome project has also demonstrated the need for both academic researchers and industrial researchers to now understand the functions of genes and gene products. The cell is the basic unit of life and it has been at the cellular level where function can be demonstrated most cost-effectively and rapidly. High content screening (HCS) was developed by Cellomics Inc. in the mid-1990s to address the need for a platform that could be used in the discovery-driven research and development required to understand the functions of genes and gene products at the level of the cell.
Download or read book New and Future Developments in Microbial Biotechnology and Bioengineering written by Ali Asghar Rastegari and published by Elsevier. This book was released on 2020-05-15 with total page 314 pages. Available in PDF, EPUB and Kindle. Book excerpt: New and Future Developments in Microbial Biotechnology and Bioengineering: Trends of Microbial Biotechnology for Sustainable Agriculture and Biomedicine Systems: Perspectives for Human Health discusses how microbial biotechnology helps us understand new strategies to reduce pathogens and drug resistance through microbial biotechnology. The most commonly used probiotic bacteria are Lactobacillus and Bifidobacterium. Therefore, the probiotic strains exhibit powerful anti-inflammatory, antiallergic and other important properties. This new book provides an indispensable reference source for engineers/bioengineers, biochemists, biotechnologists, microbiologists, pharmacologists, and researchers who want to know about the unique properties of this microbe and explore its sustainable biomedicine future applications. - Introduces the principles of microbial biotechnology and its application for sustainable biomedicine system - Explores various microbes and their beneficial application for biofortification of crops for micronutrients - Explains the potentials and significance of probiotics, prebiotics and synbiotics in health and disease - Includes current applications of beneficial microbes as Functional Food Products of Pharmaceutical Importance
Download or read book Development of a Green Fluorescent Protein Reporter as a Yeast Genotoxicity Biosensor written by Nicholas Billinton and published by . This book was released on 2000 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
