Abstract：
:Small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) are techniques used to extract structural parameters and determine the overall structures and shapes of biological macromolecules, complexes and assemblies in solution. The scattering intensities measured from a sample contain contributions from all atoms within the illuminated sample volume, including the solvent and buffer components, as well as the macromolecules of interest. To obtain structural information, it is essential to prepare an exactly matched solvent blank so that background scattering contributions can be accurately subtracted from the sample scattering to obtain the net scattering from the macromolecules in the sample. In addition, sample heterogeneity caused by contaminants, aggregates, mismatched solvents, radiation damage or other factors can severely influence and complicate data analysis, so it is essential that the samples be pure and monodisperse for the duration of the experiment. This protocol outlines the basic physics of SAXS and SANS, and it reveals how the underlying conceptual principles of the techniques ultimately 'translate' into practical laboratory guidance for the production of samples of sufficiently high quality for scattering experiments. The procedure describes how to prepare and characterize protein and nucleic acid samples for both SAXS and SANS using gel electrophoresis, size-exclusion chromatography (SEC) and light scattering. Also included are procedures that are specific to X-rays (in-line SEC-SAXS) and neutrons, specifically preparing samples for contrast matching or variation experiments and deuterium labeling of proteins.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Jeffries CM,Graewert MA,Blanchet CE,Langley DB,Whitten AE,Svergun DIdoi
10.1038/nprot.2016.113subject
Has Abstractpub_date
2016-11-01 00:00:00pages
2122-2153issue
11eissn
1754-2189issn
1750-2799pii
nprot.2016.113journal_volume
11pub_type
杂志文章相关文献
Nature Protocols文献大全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：:Chromatin and transcriptional processes are among the most intensively studied fields of biology today. The introduction of chromatin immunoprecipitations (ChIP) represents a major advancement in this area. This powerful method allows researchers to probe specific protein-DNA interactions in vivo and to estimate the d...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.27
更新日期：2006-01-01 00:00:00
abstract：:The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody-secreting plasmablasts can be enriched in vivo, in a severe combined immunode...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2014.104
更新日期：2014-07-01 00:00:00
abstract：:This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcr...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2017.148
更新日期：2018-05-01 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：: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：: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：:Fast, high-resolution mapping of heterogeneous interfaces with a wide elastic modulus range is a major goal of atomic force microscopy (AFM). This goal becomes more challenging when the nanomechanical mapping involves biomolecules in their native environment. Over the years, several AFM-based methods have been develop...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-018-0070-1
更新日期：2018-12-01 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：:Carbohydrate microarrays have received considerable attention as an advanced technology for the rapid analysis of carbohydrate-protein interactions. This protocol provides detailed procedures for the preparation of carbohydrate microarrays by immobilizing hydrazide-conjugated carbohydrates on epoxide-derivatized glass...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.373
更新日期：2007-01-01 00:00:00
abstract：:Traumatic brain injury (TBI) is a major cause of mortality and morbidity. Various attempts have been made to replicate clinical TBI using animal models. The fluid-percussion model (FP) is one of the oldest and most commonly used models of experimentally induced TBI. Both central (CFP) and lateral (LFP) variations of t...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2010.112
更新日期：2010-09-01 00:00:00
abstract：:The genomic instability of stem cells in culture, caused by their routine in vitro propagation or by their genetic manipulation, is deleterious both for their clinical application and for their use in basic research. Frequent evaluation of the genomic integrity of stem cells is thus required, and it is usually perform...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2013.051
更新日期：2013-05-01 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 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：:Periodontal disease (PD) is a common dental disease associated with the interaction between dysbiotic oral microbiota and host immunity. It is a prevalent disease, resulting in loss of gingival tissue, periodontal ligament, cementum and alveolar bone. PD is a major form of tooth loss in the adult population. Experimen...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-018-0035-4
更新日期：2018-10-01 00:00:00
abstract：:Collagen-mimetic peptides and lipopeptides are widely used as substrates for matrix degrading enzymes, as new biomaterials for tissue engineering, as drug delivery systems and so on. However, the preparation and subsequent purification of these peptides and their fatty-acid conjugates are really challenging. Herein, w...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2009.195
更新日期：2010-01-01 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：:In recent years, many mouse models have been developed to mark and trace the fate of adult cell populations using fluorescent proteins. High-resolution visualization of such fluorescent markers in their physiological setting is thus an important aspect of adult stem cell research. Here we describe a protocol to produc...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2011.365
更新日期：2011-07-28 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：:The glycocalyx comprises glycosylated proteins and lipids and fcorms the outermost layer of cells. It is involved in fundamental inter- and intracellular processes, including non-self-cell and self-cell recognition, cell signaling, cellular structure maintenance, and immune protection. Characterization of the glycocal...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-020-0350-4
更新日期：2020-08-01 00:00:00
abstract：:This protocol outlines the carboxyfluorescein diacetate succinimidyl ester (CFSE) method for following the proliferation of human lymphocytes in vitro and mouse lymphocytes both in vitro and in vivo. The method relies on the ability of CFSE to covalently label long-lived intracellular molecules with the highly fluores...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.296
更新日期：2007-01-01 00:00:00
abstract：:Metabolic labeling of glycans with a bioorthogonal chemical reporter such as the azide enables their visualization in cells and organisms as well as the enrichment of specific glycoprotein types for proteomic analysis. This process involves two steps. Azido sugars are fed to cells or organisms and integrated by the gl...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.422
更新日期：2007-01-01 00:00:00
abstract：:Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in Escherichia coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraint...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2008.227
更新日期：2009-01-01 00:00:00
abstract：:Gram-negative and Gram-positive bacteria release a variety of membrane vesicles through different formation routes. Knowledge of the structure, molecular cargo and function of bacterial extracellular vesicles (BEVs) is primarily obtained from bacteria cultured in laboratory conditions. BEVs in human body fluids have b...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-019-0236-5
更新日期：2020-01-01 00:00:00
abstract：:Limbal stem cells (LSCs) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. In this protocol, we describe a method of es...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2010.115
更新日期：2010-08-01 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：:Single-cell electroporation allows transfection of plasmid DNA or macromolecules into individual living cells using modified patch electrodes and common electrophysiological equipment. This protocol is optimized for rapid in vivo electroporation of Xenopus laevis tadpole brains with DNA, dextrans, morpholinos and comb...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.186
更新日期：2006-01-01 00:00:00
abstract：:High throughput microarray transcription analyses provide us with the expression profiles for large amounts of plant genes. However, their tissue and cellular resolution is limited. Thus, for detailed functional analysis, it is still necessary to examine the expression pattern of selected candidate genes at a cellular...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.333
更新日期：2006-01-01 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：:Multiplex-fluorescence in situ hybridization (M-FISH) was initially developed to stain human chromosomes--the 22 autosomes and X and Y sex chromosomes--with uniquely distinctive colors to facilitate karyotyping. The characteristic spectral signatures of all different combinations of fluorochromes are determined by mul...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.160
更新日期：2006-01-01 00:00:00
