Real-time cell assays of phospholipase A(2)s using fluorogenic phospholipids.

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

authors： Fischhaber PL,McDaniel LD,Friedberg EC

更新日期：2006-01-01 00:00:00

abstract：:β-arrestins (βarrs) are multifunctional proteins that interact with activated and phosphorylated G protein-coupled receptors (GPCRs) to regulate their signaling and trafficking. Understanding the intricate details of GPCR-βarr interaction continues to be a key research area in the field of GPCR biology. Bimane fluores...

journal_title：Methods in enzymology

authors： Srivastava A,Baidya M,Dwivedi-Agnihotri H,Shukla AK

更新日期：2020-01-01 00:00:00

abstract：:The anaphase-promoting complex (APC) is a central regulator of the eukaryotic cell cycle and functions as an E3 ubiquitin protein ligase to catalyze the ubiquitination of a number of cell cycle regulatory proteins. The APC contains at least 13 subunits in addition to two activator subunits, Cdc20 and Cdh1, that associ...

journal_title：Methods in enzymology

authors： Passmore LA,Barford D,Harper JW

更新日期：2005-01-01 00:00:00

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...

journal_title：Methods in enzymology

authors： Locker N,Lukavsky PJ

更新日期：2007-01-01 00:00:00

abstract：:Many methods have been developed for the cloning of PCR products. These methods include blunt-end cloning, TA cloning, and using restriction sites incorporated into the PCR primers. The restrictionless cloning technique allows efficient directional cloning of PCR products into any cloning site within a vector regardle...

journal_title：Methods in enzymology

authors： Algire MA

更新日期：2013-01-01 00:00:00

abstract：:kappaB-Ras1 and kappaB-Ras2 are two small proteins that display similarity at the amino acid level to Ras-like small GTPases. Although little is known about the function of the kappaB-Ras proteins, they have been shown to interfere with activation of transcription factor NF-kappaB. They accomplish this by binding to I...

journal_title：Methods in enzymology

authors： Huxford T,Ghosh G

更新日期：2006-01-01 00:00:00

abstract：:Guanine nucleotide exchange factors (GEFs) are essential regulators of the spatiotemporal conditions of small GTP-binding protein (SMG) activation. Their cellular activities combine the biochemical stimulation of GDP/GTP exchange, which leads to the active conformation of the SMG, to the detection of upstream signals ...

journal_title：Methods in enzymology

authors： Zeeh JC,Antonny B,Cherfils J,Zeghouf M

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

authors： Sun J,Li H

更新日期：2010-01-01 00:00:00

abstract：:ClpB belongs to the Hsp100 family of ring-forming heat-shock proteins involved in degradation of unfolded/misfolded proteins and in reactivation of protein aggregates. ClpB monomers reversibly associate to form the hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stres...

journal_title：Methods in enzymology

authors： Alfonso C,del Castillo U,Martín I,Muga A,Rivas G

更新日期：2015-01-01 00:00:00

abstract：:Tubulin is known to form high-affinity complexes with certain G proteins. The formation of such complexes allows tubulin to activate Galpha and fosters a system whereby elements of the cytoskeleton can influence G-protein signaling. This article describes the interaction between tubulin and G proteins and discusses me...

journal_title：Methods in enzymology

authors： Rasenick MM,Donati RJ,Popova JS,Yu JZ

更新日期：2004-01-01 00:00:00

abstract：:Bile acids are converted to their glycine and taurine N-acyl amidates by enzymes in the liver in a two-step process. This conjugation reaction increases the aqueous solubility of bile acids, particularly in the acidic environment of the initial portion of the small intestine. In the first step, bile acid coenzyme A (C...

journal_title：Methods in enzymology

authors： Shonsey EM,Wheeler J,Johnson M,He D,Falany CN,Falany J,Barnes S

更新日期：2005-01-01 00:00:00

abstract：:Neurons are probably the most highly polarized cell type and typically develop a single axon and several dendrites. The establishment of a polarized morphology and the functional specialization of axonal and dendritic compartments are essential steps in the differentiation of neurons. Primary cultures of dissociated h...

journal_title：Methods in enzymology

authors： Schwamborn JC,Li Y,Püschel AW

更新日期：2006-01-01 00:00:00

abstract：:Inducible expression systems represent the founding technology for the emergence of synthetic biology in mammalian cells. The core molecules in these systems are bacterial regulator proteins that bind to or dissociate from a cognate DNA operator sequence in response to an exogenous stimulus like a small-molecule induc...

journal_title：Methods in enzymology

authors： Karlsson M,Weber W,Fussenegger M

更新日期：2011-01-01 00:00:00

abstract：:In this chapter we discuss methods to study autophagic cell death. A large body of evidence demonstrates that autophagy is a cell survival mechanism in response to starvation. The role of autophagy in cell death, however, has long been controversial. Recently, molecular approaches have provided direct evidence that au...

journal_title：Methods in enzymology

authors： Lenardo MJ,McPhee CK,Yu L

更新日期：2009-01-01 00:00:00

abstract：:Quantitative real-time polymerase chain reaction (qRT-PCR) is a flexible and scalable method for analyzing transcript abundance that can be used at a single gene or high-throughput (>100 genes) level. Information obtained from this technique can be used as an indicator of potential regulation of glycosylation at the t...

journal_title：Methods in enzymology

authors： Nairn AV,dela Rosa M,Moremen KW

更新日期：2010-01-01 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：:Sulfatases hydrolyze sulfate esters on a variety of molecules including glycosaminoglycans, sulfoglycolipids, and cytosolic steroids. These enzymes are found in a wide range of organisms with their basic enzymatic mechanisms broadly conserved. In mammals, many of the sulfatases localize in the lysosome and exhibit enz...

journal_title：Methods in enzymology

authors： Uchimura K,Morimoto-Tomita M,Rosen SD

更新日期：2006-01-01 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：:Numerous aspects of the immune system operate on the basis of complex regulatory networks that are amenable to mathematical and computational modeling. Several modeling frameworks have recently been applied to simulating the immune system, including systems of ordinary differential equations, delay differential equati...

journal_title：Methods in enzymology

authors： Kim PS,Levy D,Lee PP

更新日期：2009-01-01 00:00:00

abstract：:Patient-derived xenografts are a useful tool in cancer immunology, as they allow researchers to study human cancers in vivo when starting with a relatively small amount of human tumor tissue. These models make it possible to study tumor cell-intrinsic changes that occur in response to external stimuli including cytoki...

journal_title：Methods in enzymology

authors： Cardenas JJ,Robles-Oteiza C,Politi K

更新日期：2020-01-01 00:00:00

abstract：:Protein glycosylation is one of the most important posttranslational modifications. Numerous biological functions are related to protein glycosylation. However, analytical challenges remain in the glycoprotein analysis. To overcome the challenges associated with glycoprotein analysis, many analytical techniques were d...

journal_title：Methods in enzymology

authors： Zhu R,Zacharias L,Wooding KM,Peng W,Mechref Y

更新日期：2017-01-01 00:00:00

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：: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

authors： Chung YH,Masuda S,Bauer CE

更新日期：2007-01-01 00:00:00

abstract：:The specificity of CRISPR-Cas systems for nucleic acid targets is determined by a combination of binding and cleavage. Understanding the mechanisms by which Cas proteins specifically select their targets is critical for the development of CRISPR-Cas systems for biotechnology applications. Moreover, the specificity of ...

journal_title：Methods in enzymology

authors： Wade JT

更新日期：2019-01-01 00:00:00

abstract：:Plots of free energy versus temperature are commonly called stability curves or Gibbs-Helmholtz curves, and they have proven to be extremely useful in protein folding and ligand-binding studies. Curvature in a Gibbs-Helmholtz or stability plot is indicative of a heat capacity change, and some of their primary uses in ...

journal_title：Methods in enzymology

authors： LiCata VJ,Liu CC

更新日期：2011-01-01 00:00:00

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

authors： Beck DA,Bennion BJ,Alonso DO,Daggett V

更新日期：2007-01-01 00:00:00

abstract：:Circadian rhythms generated by cell autonomous biological clocks allow for the appropriate temporal synchronization of physiology and behavior, optimizing the efficiency of biological systems. Circadian oscillators and functions have been uncovered in both central and peripheral tissues. This article describes methodo...

journal_title：Methods in enzymology

authors： Rudic RD,Curtis AM,Cheng Y,FitzGerald G

更新日期：2005-01-01 00:00:00

abstract：:Pulsed electron double resonance technique, also known as double electron-electron resonance, jointly with site-directed spin labeling (SDSL) have been used extensively for studying structures and structural change. During the last decades, significant enhancements have been made by optimization of the experimental pr...

journal_title：Methods in enzymology

authors： El Mkami H,Norman DG

更新日期：2015-01-01 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

abstract：:The Ras gene family has been implicated in the development of many human epithelial cancers. Mutations in K-ras or its downstream mediator BRAF have been detected in about two thirds of low-grade serous carcinomas and borderline serous tumors; mutations in K-ras are also often present in benign and invasive mucinous o...

journal_title：Methods in enzymology

authors： Rosen DG,Yang G,Bast RC Jr,Liu J

更新日期：2006-01-01 00:00:00