Abstract:
:This protocol describes an in vitro approach for measuring the kinetics and affinities of interactions between membrane-anchored proteins. This method is particularly established for dissecting the interaction dynamics of cytokines with their receptor subunits. For this purpose, the receptor subunits are tethered in an orientated manner onto solid-supported lipid bilayers by using multivalent chelator lipids. Interaction between the ligand with the receptor subunits was probed by a combination of surface-sensitive spectroscopic detection techniques. Label-free detection by reflectance interferometry is used for following assembly of the membrane and tethering of the receptor subunits in quantitative terms. Total internal reflection spectroscopy is used for monitoring ligand binding to the membrane-anchored receptor, for monitoring ligand-receptor interactions by FRET and for monitoring ligand-exchange kinetics. These assays can be used for determining the affinities and stabilities of ligand-receptor complexes in plane of the membrane. The techniques described in this protocol can be established in 2-3 months.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Gavutis M,Lata S,Piehler Jdoi
10.1038/nprot.2006.270subject
Has Abstractpub_date
2006-01-01 00:00:00pages
2091-103issue
4eissn
1754-2189issn
1750-2799pii
nprot.2006.270journal_volume
1pub_type
杂志文章相关文献
Nature Protocols文献大全abstract::We describe the use of a microfabricated cell culture substrate, consisting of a uniform array of closely spaced, vertical, elastomeric microposts, to study the effects of substrate rigidity on cell function. Elastomeric micropost substrates are micromolded from silicon masters comprised of microposts of different hei...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2010.189
更新日期:2011-02-01 00:00:00
abstract::Optogenetic tools provide users the ability to photocontrol the activity of cells. Commonly, activation is achieved by expression of proteins from photosynthetic organisms, for example, microbial opsins (e.g., ChR2). Alternatively, a sister approach, synthetic optogenetics, enables photocontrol over proteins of mammal...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-018-0118-2
更新日期:2019-03-01 00:00:00
abstract::This protocol describes a methodology for imaging the sequestration of infected erythrocytes of the rodent malaria parasite Plasmodium berghei in the bodies of live mice or in dissected organs, using a transgenic parasite that expresses luciferase. Real-time imaging of infected erythrocytes is performed by measuring b...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.69
更新日期:2006-01-01 00:00:00
abstract::The Buccal Micronucleus Cytome (BMCyt) assay is a minimally invasive method for studying DNA damage, chromosomal instability, cell death and the regenerative potential of human buccal mucosal tissue. This method is increasingly used in molecular epidemiological studies for investigating the impact of nutrition, lifest...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.53
更新日期:2009-01-01 00:00:00
abstract::Stabilized α-helices and nonpeptidic helix mimetics have emerged as powerful molecular scaffolds for the discovery of protein-protein interaction inhibitors. Protein-protein interactions often involve large contact areas, which are often difficult for small molecules to target with high specificity. The hypothesis beh...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2010.146
更新日期:2010-11-01 00:00:00
abstract::Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we de...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.382
更新日期:2011-09-22 00:00:00
abstract::Brachypodium distachyon is emerging as a new model system for bridging research into temperate cereal crops, such as wheat and barley, and for promoting research in novel biomass grasses. Here, we provide an adapter ligation PCR protocol that allows the large-scale characterization of T-DNA insertions into the genome ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.32
更新日期:2009-01-01 00:00:00
abstract::Cell migration through 3D extracellular matrices (ECMs) is crucial to the normal development of tissues and organs and in disease processes, yet adequate analytical tools to characterize 3D migration are lacking. The motility of eukaryotic cells on 2D substrates in the absence of gradients has long been described usin...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.030
更新日期:2015-03-01 00:00:00
abstract::We describe a protocol for easy isolation and culture of human umbilical vein endothelial cells (HUVECs) to supply every researcher with a method that can be applied in cell biology laboratories with minimum equipment. Endothelial cells (ECs) are isolated from umbilical vein vascular wall by a collagenase treatment, t...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.54
更新日期:2007-01-01 00:00:00
abstract::This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF; 1-35 kDa; a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2012.055
更新日期:2012-05-31 00:00:00
abstract::Mass spectrometry (MS)-based proteomics has become the preferred tool for the analysis of protein phosphorylation. To be successful at such an endeavor, there is a requirement for an efficient enrichment of phosphopeptides. This is necessary because of the substoichiometric nature of phosphorylation at a given site an...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.010
更新日期:2013-03-01 00:00:00
abstract::Studies on colonic cells in the lamina propria (LP) of mice are important for understanding the cellular and immune responses in the gut, especially in inflammatory bowel diseases (such as morbus crohn and colitis ulcerosa). This protocol details a method to isolate LP cells and characterize freshly isolated cells by ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.315
更新日期:2007-01-01 00:00:00
abstract::The adeno-associated virus (AAV) is one of the most promising viral vectors for human gene therapy. As with any potential therapeutic system, a thorough understanding of it at the in vitro and in vivo levels is required. Over the years, numerous methods have been developed to better characterize AAV vectors. These met...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.207
更新日期:2006-01-01 00:00:00
abstract::High-throughput sequencing of the variable domains of immune receptors (antibodies and T cell receptors (TCRs)) is of key importance in the understanding of adaptive immune responses in health and disease. However, the sequencing of both immune receptor chains (VH+VL or TCRβ/δ+TCRα/γ) at the single-cell level for typi...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2016.024
更新日期:2016-03-01 00:00:00
abstract::The goal of many current studies of neovascularization is to define the phenotype of vascular cell populations of different origins and to determine how such cells promote assembly of vascular channel. Here, we describe a protocol to immunophenotype vascular cells by high-resolution imaging and by fluorescence-activat...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.537
更新日期:2008-01-01 00:00:00
abstract::N-Succinimidyl 3-(di-tert-butyl[(18)F]fluorosilyl)benzoate ([(18)F]SiFB) is a highly reactive prosthetic group for radiolabeling of proteins for use in positron emission tomography (PET). It is similar to N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB), the 'gold-standard' prosthetic group for protein (18)F-labeli...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2012.110
更新日期:2012-11-01 00:00:00
abstract::In most organisms, one crossover (CO) event inhibits the chances of another nearby event. The term used to describe this phenomenon is 'CO interference'. Here, we describe a protocol for quickly generating large data sets that are amenable to CO interference analysis in the flowering plant, Arabidopsis thaliana. We em...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.491
更新日期:2008-01-01 00:00:00
abstract::Hypochlorous acid (HOCl) is a critical member of the reactive oxygen species (ROS) produced by immune cells to fight infections. On the other hand, HOCl in homeostasis causes oxidative damage to biomolecules and is linked to many diseases, including inflammatory, neurodegenerative, and cardiovascular diseases. Herein,...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-018-0041-6
更新日期:2018-10-01 00:00:00
abstract::Small ubiquitin-like modifier (SUMO) post-translational modification (PTM) of proteins has a crucial role in the regulation of important cellular processes. This protocol describes the chemical synthesis of functional SUMO-peptide conjugates. The two crucial stages of this protocol are the solid-phase synthesis of pep...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.013
更新日期:2015-02-01 00:00:00
abstract::This protocol describes an optimized method for direct in vitro monitoring of homo- and heterotypic axon-axon interactions involved in the developmental assembly of neural circuits. The assay exploits a classical example of heterotypic axonal interactions by modeling the sequential extension of spinal motor and somato...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.442
更新日期:2012-01-26 00:00:00
abstract::This protocol provides a detailed procedure for the chemical synthesis of proteins through native chemical ligation of peptide hydrazides. The two crucial stages of this protocol are (i) the solid-phase synthesis of peptide hydrazides via Fmoc chemistry and (ii) the native chemical ligation of peptide hydrazides throu...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.152
更新日期:2013-12-01 00:00:00
abstract::The elucidation of protein-protein interaction networks is a crucial task in the postgenomic era. In this protocol, we describe our approach to discover protein-protein interactions using the surface plasmon resonance technique coupled to mass spectrometry (MS). A peptide or a protein is immobilized on a sensor chip a...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.84
更新日期:2009-01-01 00:00:00
abstract::The new generation of synchrotrons and microfocused beamlines has enabled great progress in X-ray protein crystallography, resulting in new 3D atomic structures for proteins of high interest to the pharmaceutical industry and life sciences. It is, however, often still challenging to produce protein crystals of suffici...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2017.108
更新日期:2017-12-01 00:00:00
abstract::Mass spectrometry imaging (MSI) enables label-free spatial mapping of hundreds of biomolecules in tissue sections. This capability provides valuable information on tissue heterogeneity that is difficult to obtain using population-averaged assays. Despite substantial developments in both instrumentation and methodology...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-019-0237-4
更新日期:2019-12-01 00:00:00
abstract::To uncover the function of and interplay between the mammalian cytosine modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), new techniques and advances in current technology are needed. To this end, we have developed oxidative bisulfite sequencing (oxBS-seq), which can quantitatively locate 5mC an...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.115
更新日期:2013-10-01 00:00:00
abstract::A protocol for ester condensation between equimolar amounts of carboxylic acids and alcohols catalyzed by bulky diarylammonium pentafluorobenzenesulfonate is described. We also present procedures for the synthesis of N-(2,6-diisopropylphenyl)-N-mesitylammonium pentafluorobenzenesulfonate. The present ester condensatio...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.254
更新日期:2007-01-01 00:00:00
abstract::Improvement of cellular uptake and cellular localization is still one of the main obstacles to the development of antisense-antigene therapeutics, including peptide nucleic acid (PNA). Cell-penetrating peptides (CPPs) such as Tat peptide and polyarginine have been widely used to improve the cellular uptake of PNA and ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.92
更新日期:2006-01-01 00:00:00
abstract::Methods for installing natural and unnatural amino acids and their modifications into proteins in a benign and precise manner are highly sought-after in protein science. Here we describe a protocol for 'post-translational mutagenesis' that enables the programmed installation of protein side chains through the use of r...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2017.087
更新日期:2017-10-01 00:00:00
abstract::The Golgi apparatus undergoes extensive disassembly during mitosis and reassembly in post-mitotic daughter cells. This process has been mimicked in vitro by treating Golgi membranes with mitotic and interphase cytosol. To determine the minimal machinery that controls the morphological change, we have developed a defin...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2010.38
更新日期:2010-04-01 00:00:00
abstract::Hydroxyl radical footprinting has been widely used for studying the structure of DNA and DNA-protein complexes. The high reactivity and lack of base specificity of the hydroxyl radical makes it an excellent probe for high-resolution footprinting of DNA-protein complexes; this technique can provide structural detail th...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.72
更新日期:2008-01-01 00:00:00