Homepage / Searching for: enzyme activity in single cells

Welcome to Read Book Online

Download enzyme activity in single cells or read enzyme activity in single cells online books in PDF, EPUB and Mobi Format. Click Download or Read Online button to get enzyme activity in single cells book now. Note:! If the content not Found, you must refresh this page manually.

Enzyme Activity In Single Cells

DOWNLOAD
Author by :
Languange Used : en
Release Date : 2019-10-31
Publisher by : Academic Press

ISBN : 9780128170915

Enzyme Activity in Single Cells, Volume 628, the latest release in the Methods of Enzymology series, discusses groundbreaking cellular physiology research that is taking place in the biological sciences. Chapters in this new release cover Spatial and temporal resolution of caspase waves in single Xenopus eggs during apoptosis, Spatial and temporal organization of metabolic complexes in cells, Measuring cellular efflux and biomolecular delivery: synthetic approaches to imaging and engineering cells, Slide-based, single-cell enzyme assays, Single-cell assays using integrated continuous-flow microfluidics, High-throughput screening of single-cell lysates, Microfluidic capture of single cells for drug resistance assays, and much more. Provides the authority and expertise of leading contributors from an international board of authors Presents the latest release in the Methods in Enzymology series Includes the latest information on Enzyme Activity in Single Cells...

Microfluidic Concentration Enhanced Single Cell Enzyme Activity Assay

DOWNLOAD
Author by : Aniruddh Sarkar
Languange Used : en
Release Date : 2013
Publisher by :

ISBN : OCLC:844761440

Cells sense stimuli, process information and respond using signaling networks regulated by enzymatic activity of various proteins. Aberrations in signaling are associated with diseases such as cancer. Most current methods lack the sensitivity to measure enzymatic activity in single cells and instead measure the average of large cell populations. Cellular heterogeneity, overlooked in these methods, is widespread and relevant. Microfabricated tools are uniquely suited to single cell analysis due to the match in size scale which enables high sensitivity, high throughput measurements. In this thesis we develop a microfluidic platform for the direct measurement of enzyme activities from selected single cells without disrupting their extracellular context. We develop modules to: enhance enzyme assay sensitivity by microfluidic confinement, interface microfluidic devices with selected single cells, enable multiplexing and then integrate these modules to perform single cell assays. We first investigate electrokinetic trapping of charged biomolecules in a nanofluidic concentrator for enhancing enzyme assay sensitivity by simultaneously accumulating enzyme and substrate into a reaction plug. Non-linear enhancement of reaction kinetics in this device is predicted by a mathematical model and experimentally verified. A linear enhancement mode is developed where only the enzyme is accumulated and is reacted with substrate later in an enclosed volume defined by integrated pneumatic valves or by micro-droplets formed using an integrated droplet generator. This device is then used to perform high-throughput measurement of secreted cellular proteases. We then develop a nicrofluidic probe for lysis and capture of the contents of selected single adherent cells from standard tissue culture platforms by creating a small lysis zone at its tip using hydrodynamic confinement. The single cell lysate is then divided and mixed with different substrates and confined in small chambers for fluorimetric assays. An integrated nanofluidic concentrator enables further concentration-enhancement. We demonstrate the ability to measure, from selected single cells, the activity of kinases: Akt, MAPKAPK2, PKA and a metabolic enzyme, GAPDH - separately or simultaneously. This assay platform can correlate single cell phenotype or extracellular context to intracellular biochemical state. We present preliminary explorations of the correlation of cell morphology or local cell population density to kinase activity....

Single Cell Analysis Of Biochemical Phenotypes

DOWNLOAD
Author by : Amanda L. Richer
Languange Used : en
Release Date : 2018
Publisher by :

ISBN : OCLC:1264680620

Recent development of high throughput single cell methods has expanded our understanding of tissue heterogeneity, cell states, and developmental biology. Current single cell methods aim to understand cell function and phenotype by measuring a variety of cell features, like DNA sequence, mRNA abundance, chromatin accessibility, cell surface proteins, and histone modifications. However, cell phenotypes are regulated by a number of factors that escape the abundance measurements of current single cell methods. To directly measure cell phenotypes, I developed a method to measure enzyme activities in single cells. To this end, I designed DNA repair substrates that can be used to measure strand incision events catalyzed by endogenous DNA repair enzymes and called the method "Haircut". Haircut semi-quantitatively measures base excision repair and ribonucleotide excision repair and has been adapted to work simultaneously in a single cell mRNA sequencing experiment. Using Haircut, I measured mRNA expression and DNA repair activities in primary human immune cells and found differences in several DNA repair enzyme activities between immune cell types. Some of the repair activity measurements were supported by mRNA abundance measurements in those cell types. While other activities, especially those catalyzed by multimeric proteins, did not correlate with gene expression measurements. Additionally, I used Haircut to measure mRNA expression and DNA repair heterogeneity in immune cells from a preliminary cohort of individuals with trisomy 21 and found no differences in DNA repair between individuals with trisomy 21 and individuals with disomy 21. In summary, I developed a platform to measure enzymatic activities in single cells. I used Haircut to measure known and previously unknown differences in DNA repair across immune cell types. The platform I developed can be modified to measure other enzymatic activities and mRNA expression in thousands of single cells and can be expanded further to measure many enzymatic activities in millions of cells. Cell phenotypes are regulated by many complex mechanisms and are difficult to predict using DNA sequence or gene expression alone. Single cell analysis of biochemical phenotypes has the potential to bridge the gap between gene expression and cell function and provide direct functional readouts where other single cell methods cannot....

Molecular Biology Of The Cell

DOWNLOAD
Author by : Bruce Alberts
Languange Used : en
Release Date : 2002
Publisher by :

ISBN : 0815332181

...

Direct Quantification Of Deubiquitinating Enzyme Activity In Single Intact Cells

DOWNLOAD
Author by : Nora Safabakhsh
Languange Used : en
Release Date : 2018
Publisher by :

ISBN : OCLC:1104348124

...

Hplc In Enzymatic Analysis

DOWNLOAD
Author by : Edward F. Rossomando
Languange Used : en
Release Date : 2009-09-25
Publisher by : John Wiley & Sons

ISBN : 9780470111000

The use of High Performance Liquid Chromatography (HPLC) techniques in the study of enzymatic reactions has grown significantly since the publication of the first edition of this highly successful book: the role of enzymes in biological research has expanded; the application of HPLC and enzymes has extended to more disciplines; advances in separation techniques and instrumentation have increased the capability of HPLC; and the discovery of new enzymes has spawned new methods of analysis. High Performance Liquid Chromatography in Enzymatic Analysis, Second Edition addresses these developments in its coverage of the refinements of HPLC methods and their use in a wide range of laboratory applications. It offers the same practical approach found in the first edition, incorporates a wealth of new information into existing chapters, and adds new chapters to deal with new applications, including capillary electrophoresis, forensic chemistry, microdialysis, and the polymerase chain reaction. Topics include: * Application of HPLC to the assay of enzymatic activities * Concepts and principles of HPLC, including the latest technological advances * Concepts and principles of capillary electrophoresis (CE) * Strategy for design of an HPLC/CE system for assay of enzyme activity * Preparation of enzymatic activities from tissues and single cells * Analysis of enzymatic activities in body fluids, including chromatobiosis * HPLC for the identification of new enzymatic activities * Fundamentals of the polymerase chain reaction * HPLC in forensics * Survey of enzymatic activities assayed by the HPLC method, including many new categories * Multienzyme systems, including many new examples * HPLC in the analysis of contaminated food "It is the ability of HPLC to accomplish separations completely and rapidly that led to its original application to problems in the life sciences, particularly those related to purification. An analysis of the literature revealed that this technique was used primarily for the purification of small molecules, macromolecules such as peptides and proteins, and more recently, antibodies. This application to purification has all but dominated the use of the method, and there has been a plethora of books, symposia, and conferences on the use of HPLC for these purposes. However, it was only a matter of time before others began to look beyond and to explore the possibilities that result from the capacity to make separations quickly and efficiently." --from the preface to the First Edition Easy to read and full of practical advice and hundreds of diagrams and examples, High Performance Liquid Chromatography in Enzymatic Analysis, Second Edition is an invaluable resource for students, researchers, and laboratory workers in analytical chemistry and biochemistry, molecular biology and cell biology, and for anyone interested in keeping up with this fast-growing field....

Chemical Cytometry

DOWNLOAD
Author by : Chang Lu
Languange Used : en
Release Date : 2010-03-08
Publisher by : Wiley-VCH

ISBN : STANFORD:36105215348777

A comprehensive introduction to the novel approach for biochemical analysis at the single cell level that covers recent methods for fractionation, detection and analysis of molecules from individual cells....

Enzymes Of Molecular Biology

DOWNLOAD
Author by : Michael M. Burrell
Languange Used : en
Release Date : 1993-05-22
Publisher by : Greenwood Publishing Group

ISBN : 0896033228

Amasses key information on a broad range of enzymes commonly used as tools in molecular biology, condensing scattered sources for ease of reference, and making data accessible to nonenzymologists designing an experiment. After an overview of nucleases, 19 chapters give background on selected enzymes (nucleic acid- and protein-modifiers), discuss essential parameters for achieving optimized reactions, and provide exemplary practical procedures and protocols. Plastic comb binding. Annotation copyright by Book News, Inc., Portland, OR...

Single Molecule Enzymology

DOWNLOAD
Author by : Gregory I. Mashanov
Languange Used : en
Release Date : 2011-08-05
Publisher by : Humana Press

ISBN : 1617792624

The last fifteen years have witnessed the birth and maturation of many original methods and the development of protocols specific to single molecule measurements and their analysis, including techniques involving optical imaging, electron microscopy, optical and magnetic trapping, and developments in atomic force microscopy. In Single Molecule Enzymology: Methods and Protocols, experts in the field provide procedures which enable the extraction of detailed information about enzyme work cycles, their static and kinetic properties, and information about their location and activity within cells. The detailed volume offers practical advice on many aspects of single molecule enzymology and includes strategic overviews of interconnected methods involved in sample preparation, single molecule measurements, and data analysis. Written in the highly successful Methods in Molecular BiologyTM series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and up-to-date, Single Molecule Enzymology: Methods and Protocols is intended for use within the diverse community of molecular biologists, biochemists, and biophysicists studying enzymes in detail and can be used by researchers planning their first single molecule study or to aid more experienced researchers in further developing their existing studies....

Directed Enzyme Evolution

DOWNLOAD
Author by : Frances H. Arnold
Languange Used : en
Release Date : 2008-02-02
Publisher by : Springer Science & Business Media

ISBN : 9781592593965

Directed evolution comprises two distinct steps that are typically applied in an iterative fashion: (1) generating molecular diversity and (2) finding among the ensemble of mutant sequences those proteins that perform the desired fu- tion according to the specified criteria. In many ways, the second step is the most challenging. No matter how cleverly designed or diverse the starting library, without an effective screening strategy the ability to isolate useful clones is severely diminished. The best screens are (1) high throughput, to increase the likelihood that useful clones will be found; (2) sufficiently sen- tive (i. e. , good signal to noise) to allow the isolation of lower activity clones early in evolution; (3) sufficiently reproducible to allow one to find small improvements; (4) robust, which means that the signal afforded by active clones is not dependent on difficult-to-control environmental variables; and, most importantly, (5) sensitive to the desired function. Regarding this last point, almost anyone who has attempted a directed evolution experiment has learned firsthand the truth of the dictum “you get what you screen for. ” The protocols in Directed Enzyme Evolution describe a series of detailed p- cedures of proven utility for directed evolution purposes. The volume begins with several selection strategies for enzyme evolution and continues with assay methods that can be used to screen enzyme libraries. Genetic selections offer the advantage that functional proteins can be isolated from very large libraries s- ply by growing a population of cells under selective conditions....