Abstract:
:Hydrogen peroxide (H2O2) is a ubiquitous biological molecule whose wide range of biological functions depends on its concentration. In this chapter, we compare the delivery of H2O2 to cells as (1) a single initial dose (bolus addition); (2) a continuous source using, for example, glucose oxidase; and (3) a steady state, in which H2O2 concentration is kept constant during the assay. Both the bolus addition and the use of a continuous source of H2O2 have as outcome concentration profiles of H2O2 that are dependent on experimental conditions and that are difficult to reproduce from the information that is usually revealed in the experimental section of most research articles. On the other hand, the outcome of delivering H2O2 as a steady state is a concentration profile that is independent of experimental conditions. The implementation of the steady state starts with the determination of the kinetics of H2O2 consumption in the system under study. Then, the amount of glucose oxidase needed to produce H2O2 at a rate that matches the rate of its consumption by cells at the desired H2O2 steady-state concentration is calculated. The setup of the steady state is initiated by adding this amount of glucose oxidase simultaneously with the desired concentration of H2O2. Because H2O2 consumption and delivery rates are matched, the initial H2O2 concentration added is kept constant during the assay. Detailed explanations on how to implement the steady state, including H2O2 measurements and adjustments in the amount of H2O2 or glucose oxidase during the assay, are described.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Marinho HS,Cyrne L,Cadenas E,Antunes Fdoi
10.1016/B978-0-12-405883-5.00010-7subject
Has Abstractpub_date
2013-01-01 00:00:00pages
159-73eissn
0076-6879issn
1557-7988pii
B978-0-12-405883-5.00010-7journal_volume
526pub_type
杂志文章abstract::Precise investigation and manipulation of gene function often require modulation in a controlled and dynamic manner. In this chapter, we describe the methods to apply HIT systems for drug inducible transcriptional activation or simultaneous activation and genome editing in human cells. Together with those for editing,...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.02.032
更新日期:2019-01-01 00:00:00
abstract::The properties of a collection of transposon Tn5 derivatives that generate reporter gene fusions and internal protein tags are summarized. Procedures utilizing several of the transposons for generating genes activated by Cre-loxP recombination and for creating large sequence-defined mutant libraries are described in d...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)21012-7
更新日期:2007-01-01 00:00:00
abstract::Primary mixed liver cells were isolated from rats that had been fed an amino acid (AA) diet in which natural protein was replaced with a defined mixture of pure AAs. Nitric oxide (NO) production from these cells in vitro was monitored using a nitric oxide (NO)-selective fluorescent probe, diaminofluorescein, followed ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)96045-X
更新日期:2005-01-01 00:00:00
abstract::Fluorescence-based imaging regimes require exposure of living samples under study to high intensities of focused incident illumination. An often underestimated, overlooked, or simply ignored fact in the design of any experimental imaging protocol is that exposure of the specimen to these excitation light sources must ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-391856-7.00039-1
更新日期:2012-01-01 00:00:00
abstract::This protocol describes Coomassie brilliant blue staining, one of the most common methods of detecting proteins in polyacrylamide gels (PAGE). ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-420119-4.00013-6
更新日期:2014-01-01 00:00:00
abstract::Regulator of G-protein signaling (RGS) domains bind directly to GTP-bound Galpha subunits and accelerate their intrinsic GTPase activity by up to several thousandfold. The selectivity of RGS proteins for individual Galpha subunits has been illustrated. Thus, the expression of RGS proteins can be used to inhibit signal...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(04)89005-0
更新日期:2004-01-01 00:00:00
abstract::The genome integrity of living organisms is constantly threatened by endogenous cellular metabolic processes and environmental agents. To quantify these low, physiologically relevant levels of DNA damage, a single-cell gel electrophoresis (comet) combined with strand-specific fluorescence in situ hybridization (FISH)-...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2017.03.023
更新日期:2017-01-01 00:00:00
abstract::Omics approaches have become popular in biology as powerful discovery tools, and currently gain in interest for diagnostic applications. Establishing the accurate genome sequence of any organism is easy, but the outcome of its annotation by means of automatic pipelines remains imprecise. Some protein-encoding genes ma...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.09.019
更新日期:2017-01-01 00:00:00
abstract::Autophagy is an ancient cell survival pathway that is induced by metabolic stress and that helps prevent bioenergetic failure. This pathway has emerged as a promising new target in cancer treatment, where agents that inhibit autophagic degradation have efficacy in preventing cancer and in treating resistant disease wh...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)04012-3
更新日期:2009-01-01 00:00:00
abstract::Portable, nanoparticle (NP)-enhanced enzyme sensors have emerged as powerful devices for qualitative and quantitative analysis of a variety of analytes for biomedicine, environmental applications, and pharmaceutical fields. This chapter describes a method for the fabrication of a portable, paper-based, inexpensive, ro...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.03.006
更新日期:2016-01-01 00:00:00
abstract::This chapter discusses experimental methods and protocols for the purification and preliminary characterization of DNA polymerases that are specialized for the replicative bypass (translesion DNA synthesis) of base or other types of DNA damage that typically arrest high-fidelity DNA synthesis, with particular emphasis...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)08022-0
更新日期:2006-01-01 00:00:00
abstract::PqqE is the first enzyme in the biosynthetic pathway of the redox cofactor pyrroloquinoline quinone (PQQ), catalyzing the formation of a carbon-carbon bond in the precursor peptide PqqA. PqqE is a radical S-adenosyl-l-methionine (SAM) (RS) enzyme, a family of enzymes that use the reductive cleavage of a [4Fe-4S] clust...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.04.002
更新日期:2018-01-01 00:00:00
abstract::Acetyl CoA:arylamine N-acetyltransferase (NAT; E.C. 2.3.1.5) enzymes play a key role in the metabolic activation of aromatic amine and nitroaromatic mutagens to electrophilic reactive intermediates. We have developed a system in which the activation of mutagens by recombinant human NAT2, expressed in Escherichia coli,...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)00011-X
更新日期:2005-01-01 00:00:00
abstract::In eukaryotes, determination of the subcellular location of a novel protein encoded in genomic or transcriptomic data provides useful clues as to its possible function. However, experimental localization studies are expensive and time-consuming. As a result, accurate in silico prediction of subcellular localization fr...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(09)05002-2
更新日期:2009-01-01 00:00:00
abstract::Astrocytes are the most abundant cell type in the brain and are a crucial part of solving its mysteries. Originally assumed to be passive supporting cells, astrocyte's functions are now recognized to include active modulation and information processing at the neural synapse. The full extent of the astrocyte contributi...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.02.006
更新日期:2019-01-01 00:00:00
abstract::The development of sensors that mimic the natural smell sensing mechanism and selectively recognizes the odorants remains highly challenging. Electrochemical based sensing approaches aiming at monitoring molecular recognition events between surface receptors and analytes in solution or in the gas phase, are one possib...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.04.071
更新日期:2020-01-01 00:00:00
abstract::Two-hybrid systems are one of the most popular, preferred, cost effective, and scalable in vivo genetic approaches for screening protein-protein interactions. A number of variants of yeast and bacterial two-hybrid systems exist, rendering them ideal for modern, flexible proteomics-driven studies. For mapping protein i...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.10.020
更新日期:2017-01-01 00:00:00
abstract::Polarized epithelial cells are characterized by distinct plasma membrane domains and asymmetrical distribution of cell surface proteins and lipids. In vertebrates, tight junctions act as a fence between the apical and basolateral domains. Although many of the key components of the polarity machinery have been identifi...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)06026-5
更新日期:2006-01-01 00:00:00
abstract::During the past several years, major progress has been accomplished in the production of "designer cellulosomes," artificial enzymatic complexes that were demonstrated to efficiently degrade crystalline cellulose. This progress is part of a global attempt to promote biomass waste solutions and biofuel production. In d...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-415931-0.00023-9
更新日期:2012-01-01 00:00:00
abstract::Adenoviral vectors expressing vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A(164)) offer a powerful method for elucidating the mechanisms of pathological angiogenesis and lymphangiogenesis and for evaluating the effectiveness of pro- and anti-angiogenesis therapies. When injected int...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)02803-6
更新日期:2008-01-01 00:00:00
abstract::We describe the basic principles for imaging frozen-hydrated specimens in a transmission electron microscope and provide a step-by-step guide to a new user, from starting up and aligning the microscope, to loading a cryo-grid into the specimen holder and inserting the holder into the microscope, to setting up the low ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)81010-9
更新日期:2010-01-01 00:00:00
abstract::Superoxide dismutases (SODs; EC 1.15.1.1) are a group of metalloenzymes which are essential to protect cells under aerobic conditions. In biological systems, it has been reported that SODs and other proteins are susceptible to be attacked by peroxynitrite (ONOO(-)) which can be originated from the reaction of nitric o...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)37031-6
更新日期:2008-01-01 00:00:00
abstract::White adipose tissue (WAT) is a heterogeneous tissue composed of lipid-filled adipocytes and several nonadipocyte cell populations, including endothelial, blood, uncharacterized stromal, and adipocyte precursor cells. Although lipid-filled adipocytes account for the majority of WAT volume and mass, nonadipocyte cell p...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-411619-1.00003-3
更新日期:2014-01-01 00:00:00
abstract::Membrane-initiated steroid signaling (MISS) involves rapid second messenger based intracellular signaling without coupling to transcription or translation. MISS activates important cellular signaling cascades such as mitogen-activated protein kinase (MAPK) or adenylate cyclase pathways. Despite its vital role in signa...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.02.018
更新日期:2020-01-01 00:00:00
abstract::Oxidized phospholipids play an important role in execution of the mitochondrial stage of apoptosis and clearance of apoptotic cells by macrophages. Therefore, the identification and quantification of oxidized phospholipids generated during apoptosis are very important. These can be achieved successfully by a newly dev...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)01419-5
更新日期:2008-01-01 00:00:00
abstract::Physical, chemical, and biological processes commonly discriminate among stable isotopes. Therefore, the stable isotope compositions of biomass, growth substrates, and products often carry the isotopic fingerprints of the processes that shape them. Therefore, measuring isotope fractionation by enzymes and cultures of ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-385112-3.00014-7
更新日期:2011-01-01 00:00:00
abstract::The ability to examine gene regulation in living cells has been greatly enabled by the development of chromatin immunoprecipitation (ChIP) methodology. ChIP captures a snapshot of protein-DNA interactions in vivo and has been used to study interactions in bacteria, yeast, and mammalian cell culture. ChIP conditions va...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-385120-8.00020-6
更新日期:2011-01-01 00:00:00
abstract::In human, rat, and mice, a UGT1 complex locus provides for developmental-, inducer-, and cell-specific synthesis of a family of chemical-detoxifying isozymes, UDP-glucuronosyltransferases, which prevent toxicities, mutagenesis, and/or carcinogenesis. Between 10 and 14 first exons with individual promoter elements are ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(05)00001-7
更新日期:2005-01-01 00:00:00
abstract::The technique of site-directed spin labeling (SDSL) provides unique information on biomolecules by monitoring the behavior of a stable radical tag (i.e., spin label) using electron paramagnetic resonance (EPR) spectroscopy. In this chapter, we describe an approach in which SDSL is integrated with computational modelin...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.07.007
更新日期:2015-01-01 00:00:00
abstract::Sequence analysis of synthetic peptides using Edman chemistry can be very useful for the elucidation of certain types of synthetic problems, such as residue deletions and the presence of common stable derivatives, and for following the progress of the synthesis itself. However, it can also be a relatively poor techniq...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/s0076-6879(97)89056-8
更新日期:1997-01-01 00:00:00