Abstract:
:Microscopy is a simple, direct technique for examining the morphology of cells and their organelles. Embedding cells in agarose and then in paraffin as 'cell blocks' allows for them to be processed in the same manner that tissue specimens are processed for histology. This method is advantageous because numerous sections can be cut from one cell block. Additionally, sectioning renders antigens within the nucleus and other cell organelles more accessible to antibodies. However, access to specialized equipment for histological tissue processing is required See alternative protocols for fixation of suspension cells on Preparation of Cells for Microscopy using Cytospin and for adherent cells on Preparation of Cells for Microscopy using Chamber Slides and Coverslips.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Koh CMdoi
10.1016/B978-0-12-420067-8.00018-0subject
Has Abstractpub_date
2013-01-01 00:00:00pages
249-55eissn
0076-6879issn
1557-7988pii
B978-0-12-420067-8.00018-0journal_volume
533pub_type
杂志文章abstract::Glycoproteins are a major class of glycoconjugates displaying a variety of mutual interactions between glycan and protein moieties that ultimately affect molecular organization. Modulation of the pendant glycan structures is important in tuning the functions of glycoproteins. Here we discuss structural aspects and som...
journal_title:Methods in enzymology
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abstract::In vitro assembly of eukaryotic translation initiation complexes requires purification of ribosomal subunits, eukaryotic initiation factors, and initiator tRNA from natural sources and therefore yields only limited material for functional and structural studies. In this chapter, we describe a robust, affinity chromato...
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更新日期:2007-01-01 00:00:00
abstract::The methodologies described in this chapter allow the reproducible preparation of native high-molecular-weight synthetase complexes of leuRL. These complexes have the ability to preferentially utilize extracellular leucine immediately upon transport and are likely the forms of the enzyme most important in the utilizat...
journal_title:Methods in enzymology
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abstract::Aliphatic carbon-halogen (C-X) bonds are prevalent in modern pharmaceuticals and bioactive natural products. Three distinct chemical strategies are known in Nature to generate these structural motifs. The first is via the nucleophilic substitution at a prefunctionalized electrophilic carbon center with a halide anion ...
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abstract::Gas chromatography-mass spectrometry (GC-MS) is one of the three most popular analytical platforms for metabolomics and is largely employed for the study of oncometabolism. Large volumes of data are usually generated in a GC-MS experiment, and many analytical steps are required to extract biologically relevant informa...
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abstract::Glycosaminoglycans (GAGs) are long unbranched polysaccharides, most of which are linked to a core protein to form proteoglycans. Depending on the nature of their backbone, one can discern galactosaminoglycans (chondroitin sulfate [CS] and dermatan sulfate [DS]) and glucosaminoglycans (heparan sulfate [HS], heparin, hy...
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abstract::Staphylococcus aureus is a Gram-positive major human pathogen involved in a wide range of human infectious diseases (from minor skin infections to septicemia, endocarditis or toxic shock syndrome). The treatment of S. aureus infections is very challenging due to the emergence of multiple antibiotic-resistant isolates....
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abstract::Glycosylation is a complex form of protein modification occurring in the secretory pathway. The addition of N- and O-glycans affects intracellular processes like the folding and trafficking of most glycoproteins. To better understand the impact of glycosylation in protein folding and maturation, parameters like glycos...
journal_title:Methods in enzymology
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abstract::We present an in vitro method to measure how Rab4 and other regulatory proteins affect microtubule-based organelle motility. The protocols utilize small-volume, disposable "microchambers" designed for epifluorescence, confocal, or other microscope platforms and into which microtubules, organelles, and primary and fluo...
journal_title:Methods in enzymology
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abstract::Scanning transmission electron microscopy (STEM) of unstained, freeze-dried biological macromolecules in the dark-field mode provides an image based on the number of electrons elastically scattered by the constituent atoms of the macromolecule. The image of each isolated particle provides information about the project...
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doi:10.1016/S0076-6879(08)36027-3
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abstract::Cell proliferation requires the coordination of multiple signaling pathways as well as the provision of metabolic substrates. Nutrients are required to generate such building blocks and their form of utilization differs to significant extents between malignant tissues and their nontransformed counterparts. Thus, oncog...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
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abstract::Two factors, climate change brought on by rising atmospheric CO2 levels and the accelerating shift toward renewable energy sources, have together worked to heighten interest in understanding how biological catalysts so effectively bring about the reduction of CO2 to formate, with potential applications for both biorem...
journal_title:Methods in enzymology
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doi:10.1016/bs.mie.2018.10.013
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abstract::One of the greatest advances in molecular genetics has been the application of selectable transposons in molecular biology. After 30 years of use in microbial genetics studies, transposons remain indispensable tools for the generation of null alleles tagged with selectable markers, genetic mapping, manipulation of chr...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)21002-4
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abstract::The programmed death 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) are negative regulators of T-cell immune function. Removal of these "brakes" in T cells results in increased activation of the immune system and controlling and eradicating tumor. The development of immune checkpoint inhibitors (ICI...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.10.008
更新日期:2019-01-01 00:00:00
abstract::Ras proteins are small GTPases that exhibit high-affinity binding to GDP and GTP and hydrolyze bound GTP to GDP. The intrinsic GTPase activity of Ras proteins is accelerated by GTPase activating proteins (GAPs), which act to attenuate GTPase signaling by accelerating the conversion of bound GTP to bound GDP. Tumor-ass...
journal_title:Methods in enzymology
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doi:10.1016/S0076-6879(05)07002-3
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abstract::The existence of an accurate force field (FF) model that reproduces the free-energy landscape is a key prerequisite for the simulation of biomineralization. Here, the stages in the development of such a model are discussed including the quality of the water model, the thermodynamics of polymorphism, and the free energ...
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abstract::Mathematical computer-assisted simulations of episodic hormone, substrate, or metabolite release can be accomplished using explicit algebra and high-speed microprocessors. Such simulations are useful to describe the behavior of single or multiple pulse generators, as well as the expected output of these systems. Simul...
journal_title:Methods in enzymology
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doi:10.1016/s0076-6879(94)40056-3
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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
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doi:10.1016/S0076-6879(06)22009-3
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abstract::Rarely is any solution simply solute and water. In vivo, solutes, such as proteins and nucleic acids, swim in a sea of water, salts, ions, small molecules, and lipids, not to mention other macromolecules. In vitro, virtually all solutions contain a mixture of aqueous solvents, or "cosolvents" [i.e., solvent(s) in addi...
journal_title:Methods in enzymology
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doi:10.1016/S0076-6879(07)28022-X
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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
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abstract::Mdm2 is a negative regulator of p53 activity and functions as an E3 ubiquitin ligase of p53. Inhibition of mdm2 E3 ligase activity will block ubiquitination and subsequent proteasome-mediated degradation of p53, resulting in the stabilization of p53 protein that could lead to the restoration of its tumor-suppressor ac...
journal_title:Methods in enzymology
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abstract::A synthetic ligand regulable system for gene transfer and expression has been developed in our laboratory based on mechanistic studies of steriod hormone receptor and transcriptional regulation. This gene switch system possesses most of the important features that are required for application of the system in biologic...
journal_title:Methods in enzymology
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abstract::Accurate protein structure determination by solution-state NMR is challenging for proteins greater than about 20kDa, for which extensive perdeuteration is generally required, providing experimental data that are incomplete (sparse) and ambiguous. However, the massive increase in evolutionary sequence information coupl...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.11.004
更新日期:2019-01-01 00:00:00
abstract::gamma-Glutamyltranspeptidase is a heterodimeric glycoprotein that catalyzes the transpeptidation and hydrolysis of the gamma-glutamyl group of glutathione and related compounds. It is known that the enzyme plays a role in the metabolism of glutathione and in salvaging constituents of glutathione. In the adult animal, ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(05)01025-6
更新日期:2005-01-01 00:00:00
abstract::Pulsed EPR methods for the study of drug binding to heme-thiolate enzymes such as cytochrome P450 and nitric oxide synthase are discussed. HYSCORE and ENDOR methods to measure (1)H of axial ligands of the heme group are described with illustrations of water serving as the axial ligand in the drug-free enzyme and ligan...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.07.005
更新日期:2015-01-01 00:00:00
abstract::Genetically encoded fluorescent sensors are essential tools in modern biological research, and recent advances in fluorescent proteins (FPs) have expanded the scope of sensor design and implementation. In this review we compare different sensor platforms, including Förster resonance energy transfer (FRET) sensors, flu...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2017.01.019
更新日期:2017-01-01 00:00:00
abstract::Measurement of catalysis by MnSOD using direct observation of the UV absorbance of superoxide allows determination of steady-state catalytic constants. Stabilizing superoxide in aprotic solvents such as dimethyl sulfoxide permits the use of stopped-flow spectrophotometry, although significant information is lost in th...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(02)49321-4
更新日期:2002-01-01 00:00:00
abstract::Autophagy is an extremely dynamic process that mediates the rapid degradation of intracellular components in response to different stress conditions. The autophagic response is executed by specific protein complexes, whose function is regulated by posttranslational modifications and interactions with positive and nega...
journal_title:Methods in enzymology
pub_type: 杂志文章
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更新日期:2017-01-01 00:00:00
abstract::Reviewing the circumstances that have led to the first monoclonal antibody against the disease-associated form of PrP, we consider the availability of PrP knockout mice and recombinant PrP, as well as a reliable conformational screening protocol as being important prerequisites for a successful immunization approach. ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(99)09010-2
更新日期:1999-01-01 00:00:00
abstract::Rab38 is a low-molecular-weight G-protein highly expressed in melanocytes of the skin and alveolar type II cells in the lung. A point mutation in the postulated GTP/GDP-interacting domain of Rab38 has been identified as the genetic lesion responsible for oculocutaneous albinism (OCA) in chocolate (cht) mice. Another p...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)38014-2
更新日期:2008-01-01 00:00:00