Abstract:
:Epitope tagged alpha 2-AR subtypes have been used to address a variety of cell biological questions, and the strategies used are readily applicable to all GPCR as well as other cell surface proteins. We have provided detailed protocols for successful utilization of the epitope-tagged receptor in the studies of protein localization and trafficking in epithelial cells, and the mechanisms by which this is achieved. We have also described reversible biotinytion strategies to examine agonist-dependent (and independent) receptor turnover at the cell surface.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Wozniak M,Saunders C,Schramm N,Keefer JR,Limbird LEdoi
10.1016/s0076-6879(02)43156-4subject
Has Abstractpub_date
2002-01-01 00:00:00pages
530-44eissn
0076-6879issn
1557-7988journal_volume
343pub_type
杂志文章abstract::Over the last 50 years cytochrome c has been used as a model system for studying electron transfer and protein folding processes. Recently, convincing evidence has been provided that this protein is also involved in other biological processes such as the apoptosis and α-synuclein aggregation. Numerous lines of evidenc...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)66006-7
更新日期:2009-01-01 00:00:00
abstract::5-FOA is an extremely useful reagent for the selection of Ura- cells amid a population of Ura+ cells. The selection is effective in transformation and recombination studies where loss of URA3+ is desired. A new plasmid shuffling procedure based on the 5-FOAR selection permits the recovery of conditional lethal mutatio...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(87)54076-9
更新日期:1987-01-01 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::Optical imaging of reporter molecules such as firefly luciferase has become a popular method of tracking and visualizing cells in living animals. Many biological processes involve ubiquitin ligases, which target specific proteins for destruction under specific sets of conditions. Importantly, the motifs recognized by ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)99036-8
更新日期:2005-01-01 00:00:00
abstract::The elucidation of structure-affinity relationships in EF-hand proteins requires a reliable assay of divalent ion affinity. In principle, isothermal titration calorimetry (ITC) should be capable of furnishing estimates for Ca2+- and Mg2+-binding constants in these systems. And because the method yields the binding ent...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)04210-9
更新日期:2009-01-01 00:00:00
abstract::Peptides corresponding to the calmodulin-binding domain of MLCK and other target enzymes promise to be useful tools in the study of calmodulin action. Not only will they help to elucidate the molecular interactions of calmodulin with specific target enzymes, but they should also prove to be useful calmodulin antagonis...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(87)39079-2
更新日期:1987-01-01 00:00:00
abstract::Biochemical analysis of the components of the mRNA decay machinery is crucial to understand the mechanisms of mRNA decay. The Lsm1p-7p-Pat1p complex is a key activator of decapping in the 5' to 3'-mRNA decay pathway that is highly conserved in all eukaryotes. The first step in this pathway is poly(A) shortening that i...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)02603-7
更新日期:2008-01-01 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
pub_type: 杂志文章
doi:10.1016/B978-0-12-396962-0.00008-2
更新日期:2012-01-01 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::A procedure for an in vitro signaling assay is described for the MAPK and NFkappaB pathways. The method uses a membrane-cleared lysate that contains all the soluble components required for activating these signaling cascades. The pathways can be activated by variety of molecules, including kinases, G-proteins, and E3 ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)38024-5
更新日期:2008-01-01 00:00:00
abstract::Iron-sulfur (Fe/S) proteins are involved in numerous key biological functions such as respiration, metabolic processes, protein translation, DNA synthesis, and DNA repair. The simplest types of Fe/S clusters include [2Fe-2S], [3Fe-4S], and [4Fe-4S] forms that sometimes are present in multiple copies. De novo assembly ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2017.11.034
更新日期:2018-01-01 00:00:00
abstract::The ability to obtain large amounts of genomic sequence for organisms and high throughput technology has led to a change in the thrust of research at the level of chromosomes in animals. In the past chromosomal analysis of animals was focused on gross changes such as inversions, translocations, and deletions for both ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)95025-8
更新日期:2005-01-01 00:00:00
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
pub_type: 杂志文章
doi:10.1016/s0076-6879(94)40056-3
更新日期:1994-01-01 00:00:00
abstract::Molecular mechanisms of dark-to-light state transitions in flavoprotein photoreceptors have been the subject of intense investigation. Blue-light sensing flavoproteins fall into three general classes that share aspects of their activation processes: LOV domains, BLUF proteins, and cryptochromes. In all cases, light-in...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.03.023
更新日期:2019-01-01 00:00:00
abstract::Extracellular thiol/disulfide redox environments are highly regulated in healthy individuals and become oxidized in disease. This oxidation affects the function of cell surface receptors, ion channels, and structural proteins. Downstream signaling due to changes in extracellular redox potential can be studied using a ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)74010-6
更新日期:2010-01-01 00:00:00
abstract::RNA-binding proteins (RBPs) coordinate post-transcriptional control of gene expression, often through sequence-specific recognition of primary transcripts or mature messenger RNAs. Hundreds of RBPs are encoded in the human genome, most with undefined or incompletely defined biological roles. Understanding the function...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.02.007
更新日期:2015-01-01 00:00:00
abstract::In this chapter, we motivate the need for photoactivatable NO donor molecules and give a brief survey of the existing chemical tools in the field. We then provide detailed protocols for the synthesis and validation of a near-infrared light-activated NO donor molecule, photoNOD-1, developed in our research group. With ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.05.003
更新日期:2020-01-01 00:00:00
abstract::The monitoring of trace amounts of nitric oxide (NO) is of great interest for biomedical applications. High-resolution infrared spectroscopy, like laser magnetic resonance spectroscopy (LMRS), is a versatile technique for the quantitative and isotope-selective analysis of low concentrations of NO. The ability to disti...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)96005-9
更新日期:2005-01-01 00:00:00
abstract::Myoglobin has recently emerged as a versatile metalloprotein scaffold for the design of efficient and selective biocatalysts for abiological carbene transfer reactions, including asymmetric cyclopropanation reactions. Over the past few years, our group has explored several strategies to modulate the carbene transfer r...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.07.007
更新日期:2020-01-01 00:00:00
abstract::Adiponectin is a circulating bioactive hormone secreted by adipocytes as oligomers ranging in size from 90 kDa trimers and 180 kDa hexamers to larger high molecular weight oligomers that may reach 18- or 36-mers in size. While total circulating adiponectin levels correlate well with metabolic health, it is the relativ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-411619-1.00013-6
更新日期:2014-01-01 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::In actively growing cells, the rate of translation initiation is relatively rapid. As a result, multiple ribosomes simultaneously engaged in translation become spaced along single mRNA molecules. These structures, termed polysomes, can be separated from ribosomal subunits and single ribosomes because they migrate fast...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-420037-1.00008-7
更新日期:2013-01-01 00:00:00
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
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)08022-0
更新日期:2006-01-01 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
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::We describe a method for molecular confinement and single-fluorophore sensitive measurement in aqueous nanodroplets in oil. The sequestration of individual molecules in droplets has become a useful tool in genomics and molecular evolution. Similarly, the use of single fluorophores, or pairs of fluorophores, to study b...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)72015-2
更新日期:2010-01-01 00:00:00
abstract::Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular ani...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.04.017
更新日期:2018-01-01 00:00:00
abstract::The Ras-regulatory network is a loosely defined composition of numerous Ras family members and effector pathways that couple to critical cell-regulatory processes. Investigators are increasingly turning to RNAi-mediated inhibition of gene expression as an effective tool to help generate authentic portraits of Ras prot...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)07022-9
更新日期:2006-01-01 00:00:00
abstract::The lung is composed of two major anatomically distinct regions-the conducting airways and gas-exchanging airspaces. From a cell biology standpoint, the conducting airways can be further divided into two major compartments, the tracheobronchial and bronchiolar airways, while the alveolar regions of the lung make up th...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)19012-6
更新日期:2006-01-01 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::The unexpected discovery of graphene and especially the follow-up explosion of interest in its properties and applications marked the beginning of a new carbon era. Graphene-based nanostructured materials are highly useful because they show great promise in the field of biotechnology and biomedicine. Owing to their un...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.05.013
更新日期:2018-01-01 00:00:00