De novo design and construction of an inducible gene expression system in mammalian cells.

abstract：:Nonsense-mediated mRNA decay (NMD) is activated by exon-junction complexes (EJCs) that are located downstream of the termination codon of the substrate mRNAs. This situation can be imitated by tethering components of the EJC to the 3' untranslated region (3' UTR) of a reporter mRNA. Here we describe the detailed use o...

journal_title：Methods in enzymology

authors： Gehring NH,Hentze MW,Kulozik AE

更新日期：2008-01-01 00:00:00

abstract：:Chemokines are essential mediators of leukocyte movement in vivo. In vitro assays of leukocyte migration cannot mimic the complex interactions with other cell types and matrix needed for cells to extravasate and migrate into tissues. Therefore, in vivo strategies to study the effects and potential relevance of chemoki...

journal_title：Methods in enzymology

authors： Amaral FA,Boff D,Teixeira MM

更新日期：2016-01-01 00:00:00

abstract：:Gene manipulation serves the purpose of providing a better understanding of the function of specific genes as well as for developing novel variants of the genes of interest. The generation of knockout genes, the alteration, depletion, or enhancement of a particular gene function through the generation of specific gene...

journal_title：Methods in enzymology

authors： Stuckey S,Storici F

更新日期：2013-01-01 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

authors： Tyurin VA,Tyurina YY,Kochanek PM,Hamilton R,DeKosky ST,Greenberger JS,Bayir H,Kagan VE

更新日期：2008-01-01 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：:This protocol describes a general protein-to-protein cross-linking procedure using the water-soluble amine-reactive homobifunctional BS(3) (bis[sulfosuccinimidyl] suberate); however, the protocol can be easily adapted using other cross-linkers of similar properties. BS(3) is composed of two sulfo-NHS ester groups and ...

journal_title：Methods in enzymology

authors： Alegria-Schaffer A

更新日期：2014-01-01 00:00:00

abstract：:Hypoxia and inflammation are coincidental events in an array of diseased tissues, including chronically inflamed sites (e.g., inflammatory bowel disease, rheumatoid arthritis), growing tumors, myocardial infarcts, atherosclerotic plaques, healing wounds, and sites of bacterial infection (Murdoch et al., 2005). An unde...

journal_title：Methods in enzymology

authors： Cummins EP,Comerford KM,Scholz C,Bruning U,Taylor CT

更新日期：2007-01-01 00:00:00

abstract：:Paraquat (PQ) is a prototypical redox cycling agent commonly used experimentally to generate reactive oxygen species and oxidative stress. Recently, PQ has also come under investigation as a potential environmental neurotoxin associated with increased risk for neurodegenerative disease developing after chronic exposur...

journal_title：Methods in enzymology

authors： Drechsel DA,Patel M

更新日期：2009-01-01 00:00:00

abstract：:Tumor cells acquire distinct genetic characteristics as a means to survive and proliferate indefinitely. Changes in the genetic code can also translate in changes at the protein level, therefore creating a distinguishable signature unique for tumor cells, and absent in normal tissues. The presence of discernable moiet...

journal_title：Methods in enzymology

authors： Battaglia S

更新日期：2020-01-01 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：:Over the past 19 years, we have developed a novel myxoma virus-derived anti-inflammatory serine protease inhibitor, termed a serpin, as a new class of immunomodulatory therapeutic. This review will describe the initial identification of viral serpins with anti-inflammatory potential, beginning with preclinical analysi...

journal_title：Methods in enzymology

authors： Chen H,Zheng D,Davids J,Bartee MY,Dai E,Liu L,Petrov L,Macaulay C,Thoburn R,Sobel E,Moyer R,McFadden G,Lucas A

更新日期：2011-01-01 00:00:00

abstract：:We describe here the components and uses of two image intensifier systems. The SIT camera system is convenient, relatively inexpensive, readily adaptable to most microscopes, and reliable. It suffers from lack of resolution at high gain levels and the inability to extend its sensitivity by accumulating an image over t...

journal_title：Methods in enzymology

authors： Willingham MC,Pastan IH

更新日期：1983-01-01 00:00:00

abstract：:By far the highest resolution of all separation techniques for intact proteins in a single analytical run continues to be by the combination of isoelectric focusing (IEF) and SDS-PAGE, originally introduced by O'Farrell [(1975). J. Biol. Chem.250, 4007-4021]. This analytical platform has seen a number of significant a...

journal_title：Methods in enzymology

authors： Friedman DB,Hoving S,Westermeier R

更新日期：2009-01-01 00:00:00

abstract：:Linkers play essential roles in the engineering of fusion proteins, and have been extensively demonstrated to affect protein properties such as expression level, solubility, and biological functions. For linker design and optimization, one of the key factors is the flexibility or rigidity of linkers, which describes t...

journal_title：Methods in enzymology

authors： Huang Z,Zhang C,Xing XH

更新日期：2021-01-01 00:00:00

abstract：:Extracellular signals dictate the biological processes of neural stem cells (NSCs) both in vivo and in vitro. The intracellular response elicited by these signals is dependent on the context in which the signal is received, which in turn is decided by previous and concurrent signals impinging on the cell. A synthesis ...

journal_title：Methods in enzymology

authors： Rajan P,Snyder E

更新日期：2006-01-01 00:00:00

abstract：:Throughout the history of single-particle electron microscopy (EM), automated technologies have seen varying degrees of emphasis and development, usually depending upon the contemporary demands of the field. We are currently faced with increasingly sophisticated devices for specimen preparation, vast increases in the ...

journal_title：Methods in enzymology

authors： Lyumkis D,Moeller A,Cheng A,Herold A,Hou E,Irving C,Jacovetty EL,Lau PW,Mulder AM,Pulokas J,Quispe JD,Voss NR,Potter CS,Carragher B

更新日期：2010-01-01 00:00:00

abstract：:The circadian clock exists to synchronize inner physiology with the external world, allowing life to anticipate and adapt to the continual changes that occur in an organism's environment. The clock architecture is highly conserved, present in almost all major branches of life. Within eukaryotes, the filamentous fungus...

journal_title：Methods in enzymology

authors： Hurley J,Loros JJ,Dunlap JC

更新日期：2015-01-01 00:00:00

abstract：:The Omega class of cytosolic glutathione transferases was initially recognized by bioinformatic analysis of human sequence databases, and orthologous sequences were subsequently discovered in mouse, rat, pig, Caenorhabditis elegans, Schistosoma mansoni, and Drosophila melanogaster. In humans and mice, two GSTO genes h...

journal_title：Methods in enzymology

authors： Whitbread AK,Masoumi A,Tetlow N,Schmuck E,Coggan M,Board PG

更新日期：2005-01-01 00:00:00

abstract：:Complex synthetic schemes catalyzed by multienzyme systems immobilized on solid materials are gaining momentum in chemical biomanufacturing. These systems harness the high chemo-, regio-, and stereoselectivity of the enzymes and the recyclability of the heterogeneous catalysts. Moreover, when the enzymes become part o...

journal_title：Methods in enzymology

authors： López-Gallego F

更新日期：2019-01-01 00:00:00

abstract：:The JNKs (c-Jun N-terminal protein kinases) play important roles in a variety of physiological and pathological functions including induction of apoptosis. A major pathway by which JNKs are activated in response to apoptotic stimuli includes the GTP*Rac1-binding scaffold protein POSH (plenty of SH3s). POSH acts as a s...

journal_title：Methods in enzymology

authors： Xu Z,Greene LA

更新日期：2006-01-01 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：:Endosomes are isolated from rat liver using high-speed centrifugation through sucrose density gradients. They are distinguishable from Golgi elements, with which they coisolate, by their capacity to concentrate internalized protein ligands (viz., insulin and epidermal growth factor (EGF)) in receptor-bound intact form...

journal_title：Methods in enzymology

authors： Posner BI,Bergeron JJ

更新日期：2014-01-01 00:00:00

abstract：:Fertilization by intracytoplasmic sperm injection (ICSI) is a powerful technique that can be used to understand better the biology of fertilization, in addition to a form of assisted reproduction both in humans and in endangered species. Mouse is often the model organism of choice to study mammalian fertilization. The...

journal_title：Methods in enzymology

authors： Stein P,Schultz RM

更新日期：2010-01-01 00:00:00

abstract：:Nitric oxide (NO) is a ubiquitous gas with potent biological effects, including vasodilation, neuronal signaling, and antimicrobial activity. NO is a free radical and can readily react with other molecules, in particular, iron centers and oxygen. At physiological concentrations in aqueous solutions, even in the presen...

journal_title：Methods in enzymology

authors： Laver JR,Stevanin TM,Read RC

更新日期：2008-01-01 00:00:00

abstract：:We have described a method using polyvalent peptide display on filamentous phage that can be used to identify ligands that bind to G protein beta gamma subunits. Also described is how to construct phage that have known beta gamma binding sequences fused to the coat protein to allow a competition analysis to be perform...

journal_title：Methods in enzymology

authors： Smrcka AV,Scott JK

更新日期：2002-01-01 00:00:00

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

authors： Nomura N,Nomura Y,Sato Y,Iwata S

更新日期：2019-01-01 00:00:00

abstract：:This protocol describes the expression and analysis of membrane proteins produced in yeast, as illustrated with Yarrowia lipolytica and Pichia pastoris. Step by step, we explain how to generate a yeast strain expressing the membrane protein of interest, how to prepare a membrane protein sample from yeast, and how to a...

journal_title：Methods in enzymology

authors： Claes K,Guerfal M,Callewaert N

更新日期：2015-01-01 00:00:00

abstract：:We describe methods for expressing and isolating formin proteins from a wide range of species and comparing quantitatively their effects on actin assembly. We first developed these procedures for purification of S. cerevisiae formins Bni1 and Bnr1 but have extended them to mammalian formins, including mouse mDia1 and ...

journal_title：Methods in enzymology

authors： Moseley JB,Maiti S,Goode BL

更新日期：2006-01-01 00:00:00

abstract：:A genetic interaction occurs when the combination of two mutations leads to an unexpected phenotype. Screens for synthetic genetic interactions have been used extensively to identify genes whose products are functionally related. In particular, synthetic lethal genetic interactions often identify genes that buffer one...

journal_title：Methods in enzymology

authors： Baryshnikova A,Costanzo M,Dixon S,Vizeacoumar FJ,Myers CL,Andrews B,Boone C

更新日期：2010-01-01 00:00:00

abstract：:This chapter described the preparation and fractionation of libraries of M13 phage displaying proteins as fusions to the major coat protein. High titer (10(13) pfu/ml) phage libraries can readily be generated using a single vector and the level of display surpasses that of gene III fusion phage. Since the synthetic VI...

journal_title：Methods in enzymology

authors： Roberts BL,Markland W,Ladner RC

更新日期：1996-01-01 00:00:00