Abstract：
:In this chapter, we describe the currently most advanced methods applied for the quantitative assessment of ROS homeostasis inside the mitochondrion. These techniques are of particular interest in the field of oxidative stress. After discussing the importance of quantifying mitochondrial ROS homeostasis, three major aspects of this phenomenon and the pertinent methodologies for detection are delineated in detail. First the most important methods, based on fluorimetric or spectrophotometric approaches, for the detection of mitochondrial ROS are described. Elimination of ROS generated inside the mitochondrion is another crucial mechanism that also needs to be quantified accurately to estimate the antioxidant capacity of mitochondria under specific conditions. Since ROS generation and elimination manifest in concert, there needs to exist independent methods for the estimation of the net effect. Such a sensitive biochemical marker in the mitochondrion is aconitase, a citric acid cycle enzyme which is greatly sensitive to ROS. We describe two procedures for the precise determination of aconitase activity. A few auxiliary techniques and good practices having relevance in the successful accomplishment of the more delicate approaches are also mentioned. All other relevant technical considerations including advantages/disadvantages of the various methods and the most common artifacts are also discussed.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Tretter L,Ambrus Adoi
10.1016/B978-0-12-801415-8.00012-6subject
Has Abstractpub_date
2014-01-01 00:00:00pages
199-223eissn
0076-6879issn
1557-7988pii
B978-0-12-801415-8.00012-6journal_volume
547pub_type
杂志文章abstract：:Methane production and consumption in anaerobic marine sediments is catalyzed by a series of reversible tetrahydromethanopterin (H(4)MPT)-linked C1 transfer reactions. Although many of these reactions are conserved between one-carbon compound utilizing microorganisms, two remain diagnostic for archaeal methane metabol...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-385112-3.00004-4
更新日期：2011-01-01 00:00:00
abstract：:Isothermal titration calorimetry (ITC) provides a sensitive and accurate means by which to study the thermodynamics of binding reactions. In addition, it enables label-free measurement of enzymatic reactions. The advent of extremely sensitive microcalorimeters have made it increasingly valuable as a tool for hit valid...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2015.07.023
更新日期：2016-01-01 00:00:00
abstract：:High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS ...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/S0076-6879(06)14008-2
更新日期：2006-01-01 00:00:00
abstract：:Using the expression strategies described here, we have demonstrated a model system whereby the sequential signaling events involved in cell proliferation and subsequent transformation regulated by G alpha 12 can be investigated. The model system presented here can also be used to study the temporal interrelationships...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/s0076-6879(02)44722-2
更新日期：2002-01-01 00:00:00
abstract：:An overview of resolution enhancement of conventional, field-swept, continuous-wave electron paramagnetic resonance spectra using Fourier transform-based deconvolution methods is presented. Basic steps that are involved in resolution enhancement of calculated spectra using an implementation based on complex discrete F...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2015.06.009
更新日期：2015-01-01 00:00:00
abstract：:Bispecific antibodies may be used to improve clinical efficacy by targeting two disease mechanisms for the treatment of complex human diseases in a single agent. Bispecific antibodies also hold promise for certain therapeutic applications difficult to achieve by single-targeting monospecific antibodies, such as immune...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-416039-2.00002-1
更新日期：2012-01-01 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：:Advanced glycation is one of the major pathophysiological posttranslational modifications. Under hyperglycemic conditions or oxidative stress, proteins and DNA are nonenzymatically modified by oxidative glycation and converted to advanced glycation endproducts (AGEs), which induce the loss of protein functions or apop...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-385928-0.00020-1
更新日期：2011-01-01 00:00:00
abstract：:The protein kinase C (PKC) family of isoenzymes may be a crucial player in transducing H2O2-induced signaling in a wide variety of physiological and pathophysiological processes. PKCs contain unique structural features that make them highly susceptible to oxidative modification. Depending on the site of oxidation and ...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-405881-1.00005-7
更新日期：2013-01-01 00:00:00
abstract：:Studies on DNA replication in S. pombe have provided powerful insights into the way in which the genome of this model eukaryote is replicated and how the replication process is controlled. These studies have been facilitated by the simplicity and range of methods available in this organism for physiological and geneti...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/s0076-6879(97)83036-4
更新日期：1997-01-01 00:00:00
abstract：:Electron cryo-microscopy (cryoEM) has advanced dramatically to become a viable tool for high-resolution structural biology research. The ultimate outcome of a cryoEM study is an atomic model of a macromolecule or its complex with interacting partners. This chapter describes a variety of algorithms and software to buil...
journal_title：Methods in enzymology
pub_type： 杂志文章,评审
doi：10.1016/bs.mie.2016.06.003
更新日期：2016-01-01 00:00:00
abstract：:Lymphocytes proliferate in response to several stimuli. In many situations, a rapid lymphocyte expansion, or the identification of a slow dividing cell subpopulation may be of great interest. Thus, it is necessary to perform reliable assays to study and compare lymphocyte subsets proliferation. For this purpose, carbo...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2019.05.020
更新日期：2020-01-01 00:00:00
abstract：:Hsp70 chaperones and their obligatory J-protein cochaperones function together in many cellular processes. Via cycles of binding to short stretches of exposed amino acids on substrate proteins, Hsp70/J-protein chaperones not only facilitate protein folding but also drive intracellular protein transport, biogenesis of ...
journal_title：Methods in enzymology
pub_type： 杂志文章,评审
doi：10.1016/bs.mie.2017.07.004
更新日期：2017-01-01 00:00:00
abstract：:The above methodology has been used to prepare a variety of PE-immunotoxins. These reagents are potent cytotoxic agents for cells in culture that bind the appropriate antibody and nontoxic when cells do not bind the antibody. PE-immunotoxins can be used to select for mutant cultured cells which lack the target of the ...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/s0076-6879(87)51015-1
更新日期：1987-01-01 00:00:00
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 stat...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-405883-5.00010-7
更新日期：2013-01-01 00:00:00
abstract：:Hydrogen sulfide is an endogenous gaseous mediator that plays important roles in many physiological processes in microbes, plants, and animals. This chapter focuses on the important roles of hydrogen sulfide in protecting tissues against injury, promoting the repair of damage, and downregulating the inflammatory respo...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2014.11.034
更新日期：2015-01-01 00:00:00
abstract：:Cell-free (CF) protein expression has emerged as one of the most efficient production platforms for membrane proteins. Central bottlenecks prevalent in conventional cell-based expression systems such as mistargeting, inclusion body formation, degradation as well as product toxicity can be addressed by taking advantage...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2014.12.016
更新日期：2015-01-01 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：:Dynamin I is a large GTPase enzyme required in membrane constriction and fission during multiple forms of endocytosis. The first method described here is for the rapid purification of native dynamin from peripheral membrane extracts of sheep brain using ammonium sulfate precipitation and affinity purification on recom...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/S0076-6879(05)04049-8
更新日期：2005-01-01 00:00:00
abstract：:Actinomycete bacteria of the genus Streptomyces are major producers of bioactive compounds for the biotechnology industry. They are the source of most clinically used antibiotics, as well as of several widely used drugs against common diseases, including cancer . Genome sequencing has revealed that the potential of St...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-385075-1.00021-4
更新日期：2011-01-01 00:00:00
abstract：:Once the appropriate site has been selected for the attachment of GFP to the sarcomeric protein, it is quite remarkable that the large size of the GFP molecule does not appear to interfere with the localization of the fluorescent sarcomeric proteins into the sarcomeric regions of the myofibrils. A similar approach usi...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/s0076-6879(99)02017-0
更新日期：1999-01-01 00:00:00
abstract：:PYP-phytochrome (Ppr) is a unique photoreceptor that contains a blue light-absorbing photoactive yellow protein (PYP) domain, a red light-absorbing phytochrome domain, and a histidine kinase domain. This chapter describes overexpression of Ppr in a strain of Escherichia coli that allows covalent attachment of substoic...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/S0076-6879(06)22009-3
更新日期：2007-01-01 00:00:00
abstract：:Coat protein complex II (COPII) vesicle-mediated protein export from the endoplasmic reticulum (ER) can be controlled by linear, independent motifs embedded within the cargo. ER export motifs directly interact with selective components of COPII vesicles and enhance cargo recruitment onto COPII vesicles. Moreover, ER e...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-391862-8.00010-7
更新日期：2013-01-01 00:00:00
abstract：:Significant progress has been made in discovering and cloning a host of proteins, including a range of glycoproteins. The availability of their predicted amino acid sequences provides useful information, including potential N-linked glycosylation sites. However, only a limited number of protein structures have been so...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/S0076-6879(06)15007-7
更新日期：2006-01-01 00:00:00
abstract：:Biotinylated molecules are extensively employed in bioanalytics and biotechnology. The currently available assays for quantification of biotin groups suffer from low sensitivity, low accuracy, or provide highly variable responses for different biotin derivatives. We developed a competitive binding assay in which avidi...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2019.10.028
更新日期：2020-01-01 00:00:00
abstract：:The combined use of rotating-frame relaxation methods, temperature-dependent measurements of line shapes and magnetization transfer experiments allows in favorable cases the examination in some detail of exchange processes that occur on the millisecond time scale. It is possible to determine not only the rate constant...
journal_title：Methods in enzymology
pub_type： 杂志文章,评审
doi：10.1016/s0076-6879(94)39023-1
更新日期：1994-01-01 00:00:00
abstract：:The kinetics of biomolecular systems can be quantified by calculating the stochastic rate constants that govern the biomolecular state vs time trajectories (i.e., state trajectories) of individual biomolecules. To do so, the experimental signal vs time trajectories (i.e., signal trajectories) obtained from observing i...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2016.08.021
更新日期：2016-01-01 00:00:00
abstract：:Antibody unfolding and aggregation have been gaining increasing concern as the need for pharmaceutical antibodies with high quality has increased. Assays for continuous application of severe stress to antibodies, which are typically employed to evaluate tolerance to aggregation or to understand the mechanism of aggreg...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2019.02.021
更新日期：2019-01-01 00:00:00
abstract：:Currently, over a hundred high-resolution structures of serpins are available, exhibiting a wide range of conformations. However, our understanding of serpin dynamics and conformational change is still limited, mainly due to challenges of monitoring structural changes and characterizing transient conformations using e...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-385950-1.00014-6
更新日期：2011-01-01 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
