Abstract:
:PRA2 was found to interact with the ER-localized protein VAMP-associated protein of 33 kDa or VAP-33 by a yeast two-hybrid screen. We describe here the purification of PRA2 and VAP-33 as well as an in vitro pull-down procedure to verify the interaction. PRA2 was found to form a large sodium dodecyl sulfate (SDS)-insoluble complex upon heat denaturation, resulting in significant reduction in the Western immunoblot signal. This phenomenon is specific to PRA2 and was not observed with PRA1. We also found that protein interaction with PRA2 is highly sensitive to detergent and describe a covalent cross-linking procedure for mammalian cell extracts to stabilize the PRA2-containing complex prior to membrane solubilization and immunoprecipitation.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Gougeon PY,Ngsee JKdoi
10.1016/S0076-6879(05)03069-7subject
Has Abstractpub_date
2005-01-01 00:00:00pages
799-807eissn
0076-6879issn
1557-7988pii
S0076-6879(05)03069-7journal_volume
403pub_type
杂志文章abstract::Complex RNA structures regulate many biological processes but are often too large for structure determination by nuclear magnetic resonance (NMR) methods. We determined the solution structure of domain II of the hepatitis C viral internal ribosome entry site (HCV IRES), a 25-kDa RNA, using a novel NMR approach. Conven...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)94016-0
更新日期:2005-01-01 00:00:00
abstract::The Arc (anoxic redox control) two-component system (TCS) is a complex signal transduction system that plays an important role in regulating energy metabolism at the level of transcription in bacteria. This system comprises the ArcB protein, a hybrid membrane-associated sensor kinase, and the ArcA protein, a typical r...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)71012-0
更新日期:2010-01-01 00:00:00
abstract::The long-pulse spallation source of the European Spallation Source-a facility under construction in Lund, Sweden-is well suited for macromolecular crystallography experiments. We review briefly the particular properties of the long-pulse source and the associated high-brilliance moderators from the point of view of in...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.01.005
更新日期:2020-01-01 00:00:00
abstract::Hsp70 chaperones and their obligatory J-protein cochaperones function together in many cellular processes. Via cycles of binding to short stretches of exposed amino acids on substrate proteins, Hsp70/J-protein chaperones not only facilitate protein folding but also drive intracellular protein transport, biogenesis of ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2017.07.004
更新日期:2017-01-01 00:00:00
abstract::Peptides are multidentate chiral ligands capable of coordinating different metal ions. Nowadays, they can be obtained with high yield and purity, thanks to the advances on peptide/protein chemistry as well as in equipment (peptide synthesizers). Based on the identity and length of their amino acid sequences, peptides ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.04.007
更新日期:2016-01-01 00:00:00
abstract::The methods described here concern the use specialized glassware to achieve accurate sub-micromolar to low-millimolar dioxygen concentrations specifically for the study of flavoproteins. Since the early 1960s, these methods have been passed through the lineage of flavin enzymologists almost exclusively as an oral trad...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.03.005
更新日期:2019-01-01 00:00:00
abstract::In polarized epithelial cells, the tight junction has been ascribed several functions including the regulation of the paracellular permeability, an impediment to the diffusion of molecules between the apical and basolateral domains, a site of delivery of transport vesicles for basolateral proteins, and a scaffold for ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)04029-2
更新日期:2005-01-01 00:00:00
abstract::RNA and DNA carry out diverse functions in biology including catalysis, splicing, gene regulation, and storage of genetic information. Interest has grown in understanding how nucleic acids perform such sophisticated functions given their limited molecular repertoire. RNA can fold into diverse shapes that often perturb...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-801122-5.00009-X
更新日期:2014-01-01 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
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(05)01015-3
更新日期:2005-01-01 00:00:00
abstract::Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and the most frequently diagnosed neoplasm during infancy. Despite of aggressive treatment strategies, the 5-year survival rate for metastatic disease is still less than 60% and, consequently, novel therapeutic approaches are needed. For incre...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)65012-6
更新日期:2009-01-01 00:00:00
abstract::Intrinsically disordered proteins (IDPs) sample structurally diverse ensembles. Characterizing the underlying distributions of conformations is a key step toward understanding the structural and functional properties of IDPs. One increasingly popular method for obtaining quantitative information on intramolecular dist...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.09.030
更新日期:2018-01-01 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
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)76004-3
更新日期:2010-01-01 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::Nonnative protein aggregation has been classically treated as an amorphous process occurring by colloidal coagulation kinetics and proceeding to an essentially irreversible endpoint often ascribed to a chaotic tangle of unfolded chains. However, some nonnative aggregates, particularly amyloid fibrils, exhibit ordered ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)13003-7
更新日期:2006-01-01 00:00:00
abstract::The Rcs phosphorelay, consisting of a hybrid sensor kinase, a phosphotransferase, and a response regulator, regulates a large number of bacterial functions. These include capsule production, the target originally defined for these regulators, a small regulatory RNA, and a growing list of additional genes, many of unkn...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)23016-2
更新日期:2007-01-01 00:00:00
abstract::The RNA-guided, sequence-specific endonuclease Cas9 has been widely adopted as genome engineering tool due to its efficiency and ease of use. Derived from the microbial CRISPR (clustered regularly interspaced short palindromic repeat) type II adaptive immune system, Cas9 has now been successfully engineered for genome...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-801185-0.00008-8
更新日期:2014-01-01 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
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)93027-9
更新日期:2005-01-01 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
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)22009-3
更新日期:2007-01-01 00:00:00
abstract::A direct transport route from early/recycling endosome (EE/RE) to the trans-Golgi network (TGN) is exploited by Shiga toxin (Mallard et al., 1998) and TGN38 (Ghosh et al., 1998). To facilitate the study of this pathway, both in vivo and in vitro transport assays using recombinant Shiga toxin B fragments (STxB) as prot...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)04039-5
更新日期:2005-01-01 00:00:00
abstract::Retinoic acid (RA), the biologically active metabolite of vitamin A, regulates a vast spectrum of biological processes, such as cell differentiation, proliferation, apoptosis, and morphogenesis. microRNAs (miRNAs) play a crucial role in regulating gene expression by binding to messenger RNA (mRNA) which leads to mRNA ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.02.009
更新日期:2020-01-01 00:00:00
abstract::Polarized epithelial cells are characterized by distinct plasma membrane domains and asymmetrical distribution of cell surface proteins and lipids. In vertebrates, tight junctions act as a fence between the apical and basolateral domains. Although many of the key components of the polarity machinery have been identifi...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)06026-5
更新日期:2006-01-01 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
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)01419-5
更新日期:2008-01-01 00:00:00
abstract::The findings in this article illustrate the complexity residing in the regulation of reversible S-glutathionylation of proteins, such as GAPDH. This is clearly reflected in the design of suitable experimental approaches designed to cope with the interaction of several redox-dependent factors. Clear interactions are de...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(02)48636-3
更新日期:2002-01-01 00:00:00
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...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)16005-X
更新日期:2006-01-01 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
pub_type: 杂志文章
doi:10.1016/0076-6879(89)78005-8
更新日期:1989-01-01 00:00:00
abstract::Complex I is the major entry point in the bacterial and mitochondrial respiratory chain. Structural knowledge of the enzyme is still limited because of its large size and complicated architecture. Only the structure of the hydrophilic domain of a bacterial Complex I has been solved to high resolution by X-ray crystall...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)04401-7
更新日期:2009-01-01 00:00:00
abstract::Recurrent aliphatic hydrophobic amino acids which occur in the sequence of a protein or a peptide at positions which form an axial, hydrophobic strip when the sequence is coiled as an alpha helix might stabilize coiling against hydrophobic surfaces. That effect can lead to helix formation against hydrophobic cores of ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(91)02013-y
更新日期:1991-01-01 00:00:00
abstract::The Microsealed Drug Delivery (MDD) system was developed to achieve several objectives: to maximize the bioavailability of a therapeutic agent in a target tissue; to minimize the adverse side effects of a therapeutic agent or its metabolites; to optimize the onset, rate, and duration of drug delivery; to maintain the...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(85)12036-7
更新日期:1985-01-01 00:00:00
abstract::Hybrid proteins or chimeras are generated by recombination of protein fragments. In the course of evolution, this mechanism has led to major diversification of protein folds and their functionalities. Similarly, protein engineers have taken advantage of this attractive strategy to build new proteins. Methods that use ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-394292-0.00018-7
更新日期:2013-01-01 00:00:00
abstract::Sedimentation velocity experiments measure the transport of molecules in solution under centrifugal force. Here, we describe a method for monitoring the sedimentation of very large biological molecular assemblies using the interference optical systems of the analytical ultracentrifuge. The mass, partial-specific volum...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.05.007
更新日期:2015-01-01 00:00:00