Abstract:
:At present, several assays that use radioisotope labeling to quantify the degradation of long-lived proteins have been developed to measure autophagic flux. Here, we describe a nonradioactive pulse-chase protocol using L-azidohomoalanine (AHA) labeling to quantify long-lived protein degradation during autophagy. AHA is used as a surrogate for L-methionine, and, when added to cultured cells grown in methionine-free medium, AHA is incorporated into proteins during de novo protein synthesis. After a chase period to remove short-lived proteins, autophagy is induced by starvation or other stimuli. Cells then undergo a 'click' reaction between the azide group of AHA and a fluorescently tagged alkyne probe. The AHA-containing proteins can then be detected by flow cytometry. This protocol is nonradioactive, sensitive and quantitative, and it is easy to perform. It is also applicable to various cell culture systems. The whole protocol is estimated to take 4-5 d to complete.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Wang J,Zhang J,Lee YM,Ng S,Shi Y,Hua ZC,Lin Q,Shen HMdoi
10.1038/nprot.2016.160subject
Has Abstractpub_date
2017-12-01 00:00:00pages
279-288issue
2eissn
1754-2189issn
1750-2799pii
nprot.2016.160journal_volume
12pub_type
杂志文章相关文献
Nature Protocols文献大全abstract::Association studies can focus on candidate gene(s), a particular genomic region, or adopt a genome-wide association approach, each of which has implications for marker selection. The strategy for marker selection will affect the statistical power of the study to detect a disease association and is a crucial element of...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.38
更新日期:2009-01-01 00:00:00
abstract::Large-insert BAC (bacterial artificial chromosome) and BIBAC (binary BAC) libraries are essential for modern genomics research for all organisms. We helped pioneer the BAC and BIBAC technologies, and by using them we have constructed hundreds of BAC and BIBAC libraries for different species of plants, animals, marine ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.456
更新日期:2012-02-16 00:00:00
abstract::An optimized procedure for the efficient preparation of 2-heptyl-3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal or PQS) and a diverse range of structurally related 2-alkyl-4-quinolones with biological activity is presented. The two-step synthesis begins with the formation of α-chloro ketones by the coupling o...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2012.054
更新日期:2012-05-24 00:00:00
abstract::Conventional gene expression studies analyze multiple cells simultaneously or single cells, for which the exact in vivo or in situ position is unknown. Although cellular heterogeneity can be discerned when analyzing single cells, any spatially defined attributes that underpin the heterogeneous nature of the cells cann...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2017.003
更新日期:2017-03-01 00:00:00
abstract::This protocol describes procedures for performing fluorescence resonance energy transfer (FRET) microscopy analysis by three different methods: acceptor photobleaching, sensitized emission and spectral imaging. We also discuss anisotropy and fluorescence lifetime imaging microscopy-based FRET techniques. By using the ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2012.147
更新日期:2013-02-01 00:00:00
abstract::This protocol measures externalization of aminophospholipids (APLs) to the outside of the plasma membrane using mass spectrometry (MS). APL externalization occurs in numerous events, and it is relevant for transplant medicine, immunity and cancer. In this protocol, externalized APLs are chemically modified by using a ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.163
更新日期:2014-01-01 00:00:00
abstract::Determining enantiomeric excess (e.e.) in chiral compounds is key to development of chiral catalyst auxiliaries and chiral drugs. Here we describe a sensitive and robust fluorescence-based assay for determining e.e. in mixtures of enantiomers of 1,2- and 1,3-diols, chiral amines, amino alcohols, and amino-acid esters....
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-020-0329-1
更新日期:2020-07-01 00:00:00
abstract::The bimolecular fluorescence complementation (BiFC) assay is a powerful tool for visualizing and identifying protein interactions in living cells. This assay is based on the principle of protein-fragment complementation, using two nonfluorescent fragments derived from fluorescent proteins. When two fragments are broug...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.16
更新日期:2008-01-01 00:00:00
abstract::Pseudo-afferent cervical lymph-duct cannulation in a sheep model allows large amounts of lymph cells to be collected under physiological conditions, carrying immune signaling information from the head tissues, including oro-nasal mucosae. Importantly, large quantities of dendritic cells (DCs) of several subtypes are o...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.147
更新日期:2006-01-01 00:00:00
abstract::Here we describe a protocol to dissect the peroxisomal matrix protein import pathway using a cell-free in vitro system. The system relies on a postnuclear supernatant (PNS), which is prepared from rat/mouse liver, to act as a source of peroxisomes and cytosolic components. A typical in vitro assay comprises the follow...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2016.147
更新日期:2016-12-01 00:00:00
abstract::The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen-independent microfluidic CTC-...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2014.044
更新日期:2014-03-01 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::This protocol explains how to discover functional signals in genomic sequences by detecting over- or under-represented oligonucleotides (words) or spaced pairs thereof (dyads) with the Regulatory Sequence Analysis Tools (http://rsat.ulb.ac.be/rsat/). Two typical applications are presented: (i) predicting transcription...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.98
更新日期:2008-01-01 00:00:00
abstract::Surface- and matrix-bound signals modulate stem cell fate in vivo and in vitro. This protocol enables the immobilization of a wide range of biomolecules that contain primary amino groups to different types of solid carriers, including glass substrates and standard polystyrene well plates. We describe how thin polymer ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2010.70
更新日期:2010-06-01 00:00:00
abstract::RNA sequencing (RNA-seq) has been rapidly adopted for the profiling of transcriptomes in many areas of biology, including studies into gene regulation, development and disease. Of particular interest is the discovery of differentially expressed genes across different conditions (e.g., tissues, perturbations) while opt...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.099
更新日期:2013-09-01 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::Detailed biochemical analysis of unmanipulated germinal center (GC) B cells has not been achieved. Previously, we designed and used a simple, economical and new magnetic bead separation scheme for the purification of 'untouched' mature GC and non-GC B cells from the spleens of immunized mice and reported the first bio...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.344
更新日期:2011-06-09 00:00:00
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::This protocol describes how to purify and radiolabel collagen for use as a substrate to assay collagenolytic members of the matrix metalloproteinases (MMPs). This assay measures enzymes that specifically cleave native triple helical collagen. After incubation of the MMP enzyme with the collagen substrate at 37 degrees...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.244
更新日期:2009-01-01 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::One of the challenges for modern neuroscience is to understand the rules of concerted neuronal function in vivo. This question can be addressed using noninvasive high-resolution imaging techniques like two-photon microscopy. This protocol describes a versatile approach for in vivo two-photon calcium imaging of neural ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.58
更新日期:2006-01-01 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::With the advent of massively parallel sequencing, considerable work has gone into adapting chromosome conformation capture (3C) techniques to study chromosomal architecture at a genome-wide scale. We recently demonstrated that the inactive murine X chromosome adopts a bipartite structure using a novel 3C protocol, ter...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2016.126
更新日期:2016-11-01 00:00:00
abstract::Mass spectrometry-based phosphoproteomic analysis is a powerful method for gaining a global, unbiased understanding of cellular signaling. Its accuracy and comprehensiveness stands or falls with the quality and choice of the applied phosphopeptide prefractionation strategy. This protocol covers a powerful but simple a...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.134
更新日期:2016-01-01 00:00:00
abstract::Gene tagging with fluorescent proteins is essential for investigations of the dynamic properties of cellular proteins. CRISPR-Cas9 technology is a powerful tool for inserting fluorescent markers into all alleles of the gene of interest (GOI) and allows functionality and physiological expression of the fusion protein. ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2018.042
更新日期:2018-06-01 00:00:00
abstract::Ex vivo perfusion systems offer a reliable, reproducible method for studying acute physiological responses of an organ to various environmental manipulations. Unlike in vitro culture systems, the cellular organization, compartmentalization and three-dimensional structure of ex vivo-perfused organs are maintained. Thes...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2012.144
更新日期:2013-01-01 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::Using siRNAs to genetically manipulate immune cells is important to both basic immunological studies and therapeutic applications. However, siRNA delivery is challenging because primary immune cells are often sensitive to the delivery materials and generate immune responses. We have recently developed an amphiphilic d...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-020-00418-9
更新日期:2021-01-01 00:00:00
abstract::Apoptosis plays a pivotal role in the regulation of cell turnover, and a defect or an excess of apoptosis has been implicated in several human diseases. Apoptosis is activated from an extracellular death signal, or from an internal pathway starting from the endoplasmatic reticulum or the mitochondria. To investigate t...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.397
更新日期:2007-01-01 00:00:00
abstract::Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-asso...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2014.016
更新日期:2014-02-01 00:00:00