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 and then exposed to brain extracts injected through the surface of the chip by a microfluidic system. The interactions between the immobilized ligand and the extracts can be monitored in real time. Proteins interacting with the peptide/protein are recovered, trypsinated and identified using MS. The data obtained are searched against a sequence database using the Mascot 2.1 software. Control experiments using blank sensor chips and/or randomized peptides are carried out to exclude nonspecific interactors. The protocol can be carried out in <3 days. Other methods, such as yeast two-hybrid systems or pull-down approaches followed by MS, are widely used to screen protein-protein interactions. However, as the yeast two-hybrid system requires protein interactions in the nucleus of yeast, proteins that are abundant in other compartments may not be detected. Pull-down approaches based on immunoprecipitation can be used to study endogenous proteins but they require specific antibodies. The protocol presented here does not require the specific labeling or modification of proteins.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Madeira A,Ohman E,Nilsson A,Sjögren B,Andrén PE,Svenningsson Pdoi
10.1038/nprot.2009.84subject
Has Abstractpub_date
2009-01-01 00:00:00pages
1023-37issue
7eissn
1754-2189issn
1750-2799pii
nprot.2009.84journal_volume
4pub_type
杂志文章相关文献
Nature Protocols文献大全abstract：:In this protocol, we describe cryoimmunolabeling methods for the subcellular localization of proteins and certain lipids. The methods start with chemical fixation of cells and tissue in formaldehyde (FA) and/or glutaraldehyde (GA), sometimes supplemented with acrolein. Cell and tissue blocks are then immersed in 2.3 M...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.365
更新日期：2007-01-01 00:00:00
abstract：:Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the a...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2015.009
更新日期：2015-01-01 00:00:00
abstract：:A big challenge in proteomics is the identification of cell-type-specific proteomes in vivo. This protocol describes how to label, purify and identify cell-type-specific proteomes in living mice. To make this possible, we created a Cre-recombinase-inducible mouse line expressing a mutant methionyl-tRNA synthetase (L27...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-018-0106-6
更新日期：2019-02-01 00:00:00
abstract：:Several noncanonical initial nucleotides (NCINs) have been found to cap RNAs and possibly regulate RNA stability, transcription and translation. NAD+ is one of the NCINs that has recently been discovered to cap RNAs in a wide range of species. Identification of the NAD+-capped RNAs (NAD-RNAs) could help to unveil the ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-020-0363-z
更新日期：2020-09-01 00:00:00
abstract：:Neurotransmitter release is triggered by membrane depolarization, Ca(2+) influx and Ca(2+) sensing by the release machinery, causing synaptic vesicle (SV) fusion with the plasma membrane. Interlinked is a complex membrane cycle in which vesicles are tethered to the release site, primed, fused and recycled. As many of ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2012.074
更新日期：2012-06-21 00:00:00
abstract：:This protocol and the accompanying software program called LEVER (lineage editing and validation) enable quantitative automated analysis of phase-contrast time-lapse images of cultured neural stem cells. Images are captured at 5-min intervals over a period of 5-15 d as the cells proliferate and differentiate. LEVER au...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2011.422
更新日期：2011-11-17 00:00:00
abstract：:The scototaxis (dark/light preference) protocol is a behavioral model for fish that is being validated to assess the antianxiety effects of pharmacological agents and the behavioral effects of toxic substances, and to investigate the (epi)genetic bases of anxiety-related behavior. Briefly, a fish is placed in a centra...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2009.225
更新日期：2010-02-01 00:00:00
abstract：:We present a protocol for visualizing and quantifying single mRNA molecules in mammalian (mouse and human) tissues. In the approach described here, sets of about 50 short oligonucleotides, each labeled with a single fluorophore, are hybridized to target mRNAs in tissue sections. Each set binds to a single mRNA molecul...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2013.109
更新日期：2013-09-01 00:00:00
abstract：:Protein digestion using a dedicated protease represents a key element in a typical mass spectrometry (MS)-based shotgun proteomics experiment. Up to now, digestion has been predominantly performed with trypsin, mainly because of its high specificity, widespread availability and ease of use. Lately, it has become appar...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2016.057
更新日期：2016-05-01 00:00:00
abstract：:Forebrain γ-aminobutyric acid (GABA) interneurons have crucial roles in high-order brain function via modulating network activities and plasticity, and they are implicated in many psychiatric disorders. Availability of enriched functional human forebrain GABA interneurons, especially those from people affected by GABA...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2013.106
更新日期：2013-09-01 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：:A procedure for the synthesis of N-succinimidyl 3-iodobenzoate labeled with any iodine isotope ([*I]SIB), which is an agent used in the radioiodination of proteins and peptides, from its tin precursor N-succinimidyl 3-(tri-n-butylstannyl)benzoate (STB) is described. Also included are protocols for the synthesis of an ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.99
更新日期：2006-01-01 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：:This protocol describes the primary culture of individual chromaffin cells derived by enzymatic digestion from the adrenal medulla of the bovine adrenal gland. Since the late 1970s, such cells have provided a useful model system to study neurotransmitter biosynthesis, storage and release in the catecholaminergic syste...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.136
更新日期：2007-01-01 00:00:00
abstract：:The point mutational spectrum over nearly any 75- to 250-bp DNA sequence isolated from cells, tissues or large populations may be discovered using denaturing capillary electrophoresis (DCE). A modification of the standard DCE method that uses cycling temperature (e.g., +/-5 degrees C), CyDCE, permits optimal resolutio...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2008.79
更新日期：2008-01-01 00:00:00
abstract：:Proteins are continually produced and degraded, to avoid the accumulation of old or damaged molecules and to maintain the efficiency of physiological processes. Despite its importance, protein turnover has been difficult to measure in vivo. Previous approaches to evaluating turnover in vivo have required custom labeli...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-019-0222-y
更新日期：2019-12-01 00:00:00
abstract：:This protocol describes a method for direct labeling and detection of small RNAs present in total RNA by splinted ligation. The assay uses a small RNA-specific bridge oligonucleotide to form base pairs with the small RNA and a 5'-end-radiolabeled ligation oligonucleotide. The captured small RNA is directly labeled by ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.530
更新日期：2008-01-01 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：:Postembryonic development is an important process of organismal maturation after embryonic growth. Despite key progress in recent years in understanding embryonic development via fluorescence time-lapse microscopy, comparatively less live-cell imaging of postembryonic development has been done. Here we describe a prot...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2012.128
更新日期：2012-12-01 00:00:00
abstract：:This protocol describes a simple means of measuring the starch content of plant tissues by solubilizing the starch, converting it quantitatively to glucose and assaying the glucose. Plant tissue must initially be frozen rapidly to stop metabolism, then extracted to remove free glucose. Starch is solubilized by heating...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.232
更新日期：2006-01-01 00:00:00
abstract：:Mouse embryonic stem cells (mESCs) are key tools for genetic engineering, development of stem cell-based therapies and basic research on pluripotency and early lineage commitment. However, successful derivation of germline-competent embryonic stem cell lines has, until recently, been limited to a small number of inbre...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2014.030
更新日期：2014-03-01 00:00:00
abstract：:This protocol describes a culture system in which inner-ear sensory tissue is produced from mouse embryonic stem (ES) cells under chemically defined conditions. This model is amenable to basic and translational investigations into inner ear biology and regeneration. In this protocol, mouse ES cells are aggregated in 9...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2014.100
更新日期：2014-01-01 00:00:00
abstract：:This protocol describes a directed evolution method for in vitro mutagenesis and recombination of polynucleotide sequences. The staggered extension process (StEP) is essentially a modified PCR that uses highly abbreviated annealing and extension steps to generate staggered DNA fragments and promote crossover events al...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.309
更新日期：2006-01-01 00:00:00
abstract：:Development of non-invasive and accurate methods to track cell fate after delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. In this protocol, we describe the in vivo monitoring of stem cell survival, proliferation and migration using reporter genes. We established stable ES c...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2009.100
更新日期：2009-01-01 00:00:00
abstract：:Adult stem cell-based organoid technology is a versatile tool for the generation and long-term maintenance of near-native 3D epithelial tissues in vitro. The generation of cancer organoids from primary patient material enables a range of therapeutic agents to be tested in the resulting organoid cultures. Patient-deriv...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-020-0379-4
更新日期：2020-10-01 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：:This protocol describes the efficient, generally applicable Ullmann coupling reaction of bromaminic acid with alkyl- or aryl-amines in phosphate buffer under microwave irradiation using elemental copper as a catalyst. The reaction leads to a number of biologically active compounds. As a prototypical example, the synth...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2010.63
更新日期：2010-05-01 00:00:00
abstract：:Cyclodepsipeptides are cyclic peptides in which at least one amide link on the backbone is replaced with an ester link. These natural products present a high structural diversity that corresponds to a broad range of biological activities. Therefore, they are very promising pharmaceutical candidates. Most of the cyclod...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2016.116
更新日期：2016-10-01 00:00:00
abstract：:Raman and Fourier transform IR (FTIR) microspectroscopic images of biological material (tissue sections) contain detailed information about their chemical composition. The challenge lies in identifying changes in chemical composition, as well as locating and assigning these changes to different conditions (pathology, ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2015.008
更新日期：2015-02-01 00:00:00
abstract：:RNA interference (RNAi) is an efficient method for silencing genes in cultured cells. Here we describe a simple RNAi approach for silencing genes in a cell type-specific and tissue-specific way in vivo. The approach, which mimics the means by which naturally occurring 'microRNA's are generated, uses a tissue-specific ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.260
更新日期：2006-01-01 00:00:00
