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Author:
Publisher: Academic Press
ISBN: 0128170905
Category : Science
Languages : en
Pages : 326
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Book Description
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
Author:
Publisher: Academic Press
ISBN: 0128170905
Category : Science
Languages : en
Pages : 326
Get Book Here
Book Description
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
Author: Aniruddh Sarkar
Publisher:
ISBN:
Category :
Languages : en
Pages : 91
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Book Description
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.
Author:
Publisher:
ISBN: 9780815332183
Category : Cells
Languages : en
Pages : 0
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Book Description
Author: Amanda L. Richer
Publisher:
ISBN:
Category :
Languages : en
Pages : 221
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Book Description
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.
Author: Jonathan V. Sweedler
Publisher: Springer Nature
ISBN: 107162525X
Category : Medical
Languages : en
Pages : 302
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Book Description
This volume discusses the latest techniques used in the diverse fields of single cell ‘omics and covers topics such as quantifying the single cell transcriptome; isolation of cells in nanoliter volumes for single cell proteomics measurements by nano-LC-MS/MS; and single cell protein characterization by immunoblotting. A wide range of methodologies are highlighted, ranging from high-yield chemical amplification to mass spectrometry and nanotechnology for the analysis of the chemical constituents of cells. In the Neuromethods series style, chapters include both in-depth overviews, as well as detailed protocols that provide the key advice from specialists you need to get successful results in your laboratory. Cutting-edge and comprehensive, Single Cell ‘Omics of Neuronal Cells is a valuable resource for experienced and novice researchers interested in learning more about this field and its future developments.
Author: Michael M. Burrell
Publisher: Greenwood Publishing Group
ISBN: 9780896033221
Category : Medical
Languages : en
Pages : 386
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Book Description
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
Author: Julianne Zedalis
Publisher:
ISBN: 9781947172401
Category : Biology
Languages : en
Pages : 1923
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Book Description
Biology for AP® courses covers the scope and sequence requirements of a typical two-semester Advanced Placement® biology course. The text provides comprehensive coverage of foundational research and core biology concepts through an evolutionary lens. Biology for AP® Courses was designed to meet and exceed the requirements of the College Board’s AP® Biology framework while allowing significant flexibility for instructors. Each section of the book includes an introduction based on the AP® curriculum and includes rich features that engage students in scientific practice and AP® test preparation; it also highlights careers and research opportunities in biological sciences.
Author: Ajit Varki
Publisher: CSHL Press
ISBN: 9780879696818
Category : Medical
Languages : en
Pages : 694
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Book Description
Sugar chains (glycans) are often attached to proteins and lipids and have multiple roles in the organization and function of all organisms. "Essentials of Glycobiology" describes their biogenesis and function and offers a useful gateway to the understanding of glycans.
Author: Vladimir V. Ghukasyan
Publisher: CRC Press
ISBN: 1466509996
Category : Medical
Languages : en
Pages : 404
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Book Description
From the Lab to Clinical Settings-Advances in Quantitative, Noninvasive Optical DiagnosticsNoninvasive fluorescence imaging techniques, novel fluorescent labels, and natural biomarkers are revolutionizing our knowledge of cellular processes, signaling and metabolic pathways, the underlying mechanisms for health problems, and the identification of n
Author: Erwin E. Sterchi
Publisher: Springer Science & Business Media
ISBN: 3642598161
Category : Science
Languages : en
Pages : 371
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Book Description
Following an overview on proteolytic enzyme assays, this text covers procedures on how to investigate and study proteases. It describes the use of specific restriction proteases as well as inhibitors of proteases to prevent unwanted proteolysis.
