Characterizing and measuring endocytosis of lipid-binding effectors in mammalian cells.

abstract：:When yeast are faced with irreparable DNA damage, they will first arrest in G2/M, via the DNA damage checkpoint pathway, but will subsequently adapt to that arrest and resume division. Here, we summarize assays that we have used to examine checkpoint adaptation. Specifically, we discuss the merits of inducing DNA dama...

journal_title：Methods in enzymology

authors： Toczyski DP

更新日期：2006-01-01 00:00:00

abstract：:Osmoregulation is the active control of the cellular water balance and encompasses homeostatic mechanisms crucial for life. The osmoregulatory system in the yeast Saccharomyces cerevisiae is particularly well understood. Key to yeast osmoregulation is the production and accumulation of the compatible solute glycerol, ...

journal_title：Methods in enzymology

authors： Hohmann S,Krantz M,Nordlander B

更新日期：2007-01-01 00:00:00

abstract：:Hematopoietic stem cells (HSCs) primarily reside in bone marrow, are defined by their ability to maintain blood homeostasis, and replenish themselves through self-renewal. Although HSC purification schemes vary from laboratory to laboratory, the resulting cell populations are similar, if not the same. This chapter wil...

journal_title：Methods in enzymology

authors： Lin KK,Goodell MA

更新日期：2006-01-01 00:00:00

abstract：:Circadian clocks control daily rhythms in physiology and behavior across all phyla. These rhythms are intrinsic to individual cells that must synchronize to their environment and to each other to anticipate daily events. Recent advances in recording from large numbers of cells for many circadian cycles have enabled re...

journal_title：Methods in enzymology

authors： Herzog ED,Kiss IZ,Mazuski C

更新日期：2015-01-01 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

authors： Hampel A,Tritz R

更新日期：1988-01-01 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

authors： Quan A,Robinson PJ

更新日期：2005-01-01 00:00:00

abstract：:Antimicrosomal antibodies in inflammatory liver diseases all seem to be directed against members of the cytochrome P450 family of proteins. These autoantigens seem to be genetically polymorphic, the autoantibodies are inhibitory, and the autoepitopes are generally conserved among species. Anti-P450 autoantibodies shar...

journal_title：Methods in enzymology

authors： Manns MP,Johnson EF

更新日期：1991-01-01 00:00:00

abstract：:The breakthrough discovery that double-stranded RNA of 21 nucleotides in length (referred to as short or small interfering RNA; siRNA) can trigger sequence-specific gene silencing in mammalian cells has led to the development of a powerful new approach to study gene function (Dillon et al., 2005; Dykxhoorn et al., 200...

journal_title：Methods in enzymology

authors： Janas J,Skowronski J,Van Aelst L

更新日期：2006-01-01 00:00:00

abstract：:DNA repair complexes play crucial roles in maintaining genome integrity, which is essential for the survival of an organism. The understanding of their modes of action is often obscure due to limited structural knowledge. Structural characterizations of these complexes are often challenging due to a poor protein produ...

journal_title：Methods in enzymology

authors： Sawicka M,Aramayo R,Ayala R,Glyde R,Zhang X

更新日期：2017-01-01 00:00:00

abstract：:A simple and convenient method is described to determine primary deuterium kinetic isotope effects (1°DKIEs) on reactions where the hydron incorporated into the reaction product is derived from solvent water. The 1°DKIE may be obtained by 1H NMR analyses as the ratio of the yields of H- and D-labeled products from a r...

journal_title：Methods in enzymology

authors： Amyes TL,Richard JP

更新日期：2017-01-01 00:00:00

abstract：:Cryptococcus neoformans is a yeastlike fungus that causes a lethal meningoencephalitis in a broad spectrum of immunocompromised patients and has become the most common cause of meningitis due to AIDS-related infections in Africa. Key to the development of new agents to control and prevent this infection is the identif...

journal_title：Methods in enzymology

authors： Hu G,Gibbons J,Williamson PR

更新日期：2008-01-01 00:00:00

abstract：:Mass spectrometry (MS) has gradually replaced classical methods as a major tool in protein sequencing and characterization. However, the sample preparation repertoire has not changed very much; it has just been adjusted to the needs of the new analytical method. In this chapter frequently used in-solution enzymatic di...

journal_title：Methods in enzymology

authors： Medzihradszky KF

更新日期：2005-01-01 00:00:00

abstract：:The diverse structures of polyketide natural products are reflected by the equally diverse polyketide biosynthetic enzymes, namely polyketide synthases (PKSs). Three major classes of PKSs are known-noniterative type I PKSs, iterative type II PKSs and acyl carrier protein-independent type III PKSs, each of which consis...

journal_title：Methods in enzymology

authors： Cheng YQ,Coughlin JM,Lim SK,Shen B

更新日期：2009-01-01 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

authors： Silverman DN,Nick HS

更新日期：2002-01-01 00:00:00

abstract：:MicroRNA (miRNA) genes are transcribed into long primary transcripts (pri-miRNAs) that get processed into mature miRNAs of about 22 nt in length by two different ribonuclease (RNase) III enzymes, Drosha and Dicer. Various experimental protocols have been developed and modified for genetic and biochemical analyses for ...

journal_title：Methods in enzymology

authors： Lee Y,Kim VN

更新日期：2007-01-01 00:00:00

abstract：:Ion channels open and close in response to diverse stimuli, and the molecular events underlying these processes are extensively modulated by ligands of both endogenous and exogenous origin. In the past decade, high-resolution structures of several channel types have been solved, providing unprecedented details of the ...

journal_title：Methods in enzymology

authors： Chakrapani S

更新日期：2015-01-01 00:00:00

abstract：:Malaria represents an emerging disease because of increasing parasite resistance against available drugs and because of increasing geographical distribution of the causative agent, Plasmodium falciparum. The complete genome of Plasmodium was sequenced recently, revealing that the parasite harbors only one glutathione ...

journal_title：Methods in enzymology

authors： Deponte M,Becker K

更新日期：2005-01-01 00:00:00

abstract：:Protein oxidation is implicated in atherogenesis and other inflammatory conditions. Measuring levels of chlorinated and nitrated proteins in biological matrices serves as a quantitative index of oxidative stress in vivo. One potential mechanism for oxidative stress involves myeloperoxidase, a heme protein expressed by...

journal_title：Methods in enzymology

authors： Shao B,Heinecke JW

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

authors： Church CD,Berry R,Rodeheffer MS

更新日期：2014-01-01 00:00:00

abstract：:Intraflagellar transport (IFT) involves the movement of large proteinaceous particles or trains along the length of ciliary and flagellar axonemal microtubules. The particles contain multiple copies of two protein complexes. As isolated from the flagellated model organism, Chlamydomonas reinhardtii, IFT A contains 6 d...

journal_title：Methods in enzymology

authors： Behal RH,Cole DG

更新日期：2013-01-01 00:00:00

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

authors： Zhao C,Wei S,Wang Y

更新日期：2019-01-01 00:00:00

abstract：:An optimal tool to unravel the role of a specific player within a cellular network or process requires its spatiotemporally resolved perturbation. Chemically induced dimerization (CID) by the rapamycin system has proven useful to induce protein dimerization or translocation with high spatiotemporal precision. Recently...

journal_title：Methods in enzymology

authors： Schifferer M,Feng S,Stein F,Schultz C

更新日期：2017-01-01 00:00:00

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...

journal_title：Methods in enzymology

authors： Wall JS,Simon MN,Lin BY,Vinogradov SN

更新日期：2008-01-01 00:00:00

abstract：:Posttranslational modifications (PTMs) of proteins perform crucial roles in regulating the biology of the cell. PTMs are enzymatic, covalent chemical modifications of proteins that typically occur after the translation of mRNAs. These modifications are relevant because they can potentially change a protein's physical ...

journal_title：Methods in enzymology

authors： Farley AR,Link AJ

更新日期：2009-01-01 00:00:00

abstract：:The procedures described in this chapter have enabled us to identify and characterize monoclonal antibodies and their respective anti-idiotypes. We have developed several different types of immunoassays which afford greater flexibility to the investigator, depending on the type of antibodies desired and the availabili...

journal_title：Methods in enzymology

authors： Kussie PH,Albright G,Linthicum DS

更新日期：1989-01-01 00:00:00

abstract：:Staphylococcus aureus ribonuclease III (Sa-RNase III) belongs to the enzyme family known to process double-stranded RNAs consisting of two turns of the RNA helix. Although the enzyme is thought to play a role in ribosomal RNA processing and gene regulation, the deletion of the rnc gene in S. aureus does not affect cel...

journal_title：Methods in enzymology

authors： Chevalier C,Huntzinger E,Fechter P,Boisset S,Vandenesch F,Romby P,Geissmann T

更新日期：2008-01-01 00:00:00

abstract：:There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However,...

journal_title：Methods in enzymology

authors： Onesto C,Shutes A,Picard V,Schweighoffer F,Der CJ

更新日期：2008-01-01 00:00:00

abstract：:Lead generation can be a very challenging phase of the drug discovery process. The two principal methods for this stage of research are blind screening and rational design. Among the rational or semirational design approaches, fragment-based drug discovery (FBDD) has emerged as a useful tool for the generation of lead...

journal_title：Methods in enzymology

authors： Lanter J,Zhang X,Sui Z

更新日期：2011-01-01 00:00:00

abstract：:T lymphocytes express on their surface a heterodimeric αβ receptor, called the T cell receptor (TCR), which recognizes foreign antigens. Unlike antibodies, the recognition requires both an antigenic peptide epitope and a protein encoded by the major histocompatibility complex (MHC). In contrast to conventional antibod...

journal_title：Methods in enzymology

authors： Stone JD,Chervin AS,Aggen DH,Kranz DM

更新日期：2012-01-01 00:00:00

abstract：:The number of genetically modified mouse lines has been increasing exponentially in the past few decades. In order to safeguard them from accidental loss and genetic drifting, to reduce animal housing cost, and to efficiently distribute them around the world, it is important to cryopreserve these valuable genetic reso...

journal_title：Methods in enzymology

authors： Takahashi H,Liu C

更新日期：2010-01-01 00:00:00