Chemical synthesis of proteins using peptide hydrazides as thioester surrogates.


: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 through in situ NaNO2 activation and thiolysis. This protocol may be of help in the synthesis of proteins that are not easily produced by recombinant technology and that include acid-sensitive modifications; it also does not involve the use of hazardous HF. The utility of the protocol is shown for the total synthesis of 140-aa-long α-synuclein, a protein that has an important role in the development of Parkinson's disease. The whole synthesis of the target protein α-synuclein in milligram scale takes ~30 working days.


Nat Protoc


Nature protocols


Zheng JS,Tang S,Qi YK,Wang ZP,Liu L




Has Abstract


2013-12-01 00:00:00














  • Measuring bone blood supply in mice using fluorescent microspheres.

    abstract::Fluorescent microspheres are commonly used to assess bone blood supply in large animals, but the technique is not widely used in smaller mammals, as traditional methods such as reference blood sampling, ventilation and catheterization are not easily applied. This protocol describes a viable alternative for measuring b...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Serrat MA

    更新日期:2009-01-01 00:00:00

  • Statistical analysis of cell migration in 3D using the anisotropic persistent random walk model.

    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


    authors: Wu PH,Giri A,Wirtz D

    更新日期:2015-03-01 00:00:00

  • Virtual karyotyping of pluripotent stem cells on the basis of their global gene expression profiles.

    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


    authors: Ben-David U,Mayshar Y,Benvenisty N

    更新日期:2013-05-01 00:00:00

  • Housing conditions and stimulus females: a robust social discrimination task for studying male rodent social recognition.

    abstract::Social recognition (SR) enables rodents to distinguish between familiar and novel conspecifics, largely through individual odor cues. SR tasks utilize the tendency for a male to sniff and interact with a novel individual more than a familiar individual. Many paradigms have been used to study the roles of the neuropept...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Macbeth AH,Edds JS,Young WS 3rd

    更新日期:2009-01-01 00:00:00

  • Structure determination protocol for transmembrane domain oligomers.

    abstract::The transmembrane (TM) anchors of cell surface proteins have been one of the 'blind spots' in structural biology because they are generally very hydrophobic, sometimes dynamic, and thus difficult targets for structural characterization. A plethora of examples show these membrane anchors are not merely anchors but can ...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Fu Q,Piai A,Chen W,Xia K,Chou JJ

    更新日期:2019-08-01 00:00:00

  • Proteome-wide identification of SUMO modification sites by mass spectrometry.

    abstract::The protein called 'small ubiquitin-like modifier' (SUMO) is post-translationally linked to target proteins at the ɛ-amino group of lysine residues. This 'SUMOylation' alters the behavior of the target protein, a change that is utilized to regulate diverse cellular processes. Understanding the target-specific conseque...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Tammsalu T,Matic I,Jaffray EG,Ibrahim AF,Tatham MH,Hay RT

    更新日期:2015-09-01 00:00:00

  • Synthesis of complex head-to-side-chain cyclodepsipeptides.

    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


    authors: Pelay-Gimeno M,Albericio F,Tulla-Puche J

    更新日期:2016-10-01 00:00:00

  • Spatial transcriptomic analysis of cryosectioned tissue samples with Geo-seq.

    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


    authors: Chen J,Suo S,Tam PP,Han JJ,Peng G,Jing N

    更新日期:2017-03-01 00:00:00

  • Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro.

    abstract::The fabrication of 3D tissues retaining the original functions of tissues/organs in vitro is crucial for optimal tissue engineering and regenerative medicine. The fabrication of 3D tissues also contributes to the establishment of in vitro tissue/organ models for drug screening. Our laboratory has developed a fabricati...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Haraguchi Y,Shimizu T,Sasagawa T,Sekine H,Sakaguchi K,Kikuchi T,Sekine W,Sekiya S,Yamato M,Umezu M,Okano T

    更新日期:2012-04-05 00:00:00

  • Construction of BIBAC and BAC libraries from a variety of organisms for advanced genomics research.

    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


    authors: Zhang HB,Scheuring CF,Zhang M,Zhang Y,Wu CC,Dong JJ,Li Y

    更新日期:2012-02-16 00:00:00

  • Transformation of the rodent malaria parasite Plasmodium chabaudi.

    abstract::The rodent malaria parasite Plasmodium chabaudi chabaudi shares many features with human malaria species, including P. falciparum, and is the in vivo model of choice for many aspects of malaria research in the mammalian host, from sequestration of parasitized erythrocytes, to antigenic variation and host immunity and ...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Spence PJ,Cunningham D,Jarra W,Lawton J,Langhorne J,Thompson J

    更新日期:2011-04-01 00:00:00

  • Design of a functional cyclic HSV1-TK reporter and its application to PET imaging of apoptosis.

    abstract::Positron emission tomography (PET) is a sensitive and noninvasive imaging method that is widely used to explore molecular events in living subjects. PET can precisely and quantitatively evaluate cellular apoptosis, which has a crucial role in various physiological and pathological processes. In this protocol, we descr...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Wang Z,Wang F,Hida N,Kiesewetter DO,Tian J,Niu G,Chen X

    更新日期:2015-05-01 00:00:00

  • Isolation of DNA from small amounts of elephant ivory.

    abstract::This protocol describes a method for the extraction of DNA from elephant ivory. These techniques are being used to assign geographic origin to poached ivory by comparing the ivory genotype to a geographic-based gene frequency map, developed separately. The method has three components: ivory pulverization, decalcificat...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Mailand C,Wasser SK

    更新日期:2007-01-01 00:00:00

  • Generation of human antral and fundic gastric organoids from pluripotent stem cells.

    abstract::The human stomach contains two primary domains: the corpus, which contains the fundic epithelium, and the antrum. Each of these domains has distinct cell types and functions, and therefore each presents with unique disease pathologies. Here, we detail two protocols to differentiate human pluripotent stem cells (hPSCs)...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Broda TR,McCracken KW,Wells JM

    更新日期:2019-01-01 00:00:00

  • Synthesis, encapsulation, purification and coupling of single quantum dots in phospholipid micelles for their use in cellular and in vivo imaging.

    abstract::A detailed protocol for the synthesis of core/shell semiconductor nanocrystal, their encapsulation into phospholipid micelles, their purification and their coupling to a controlled number of small molecules is given. The protocol for the core/shell quantum dot (QD) CdSe/CdZnS synthesis has been specifically designed w...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Carion O,Mahler B,Pons T,Dubertret B

    更新日期:2007-01-01 00:00:00

  • Orthotopic mouse lung transplantation as experimental methodology to study transplant and tumor biology.

    abstract::Unlike transplantation of other solid organs, vascularized mouse lung transplantation has only recently been developed. In this protocol, we describe a detailed method for performing a vascularized and aerated mouse orthotopic lung transplant, which to date represents the most physiological mouse model of lung transpl...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Krupnick AS,Lin X,Li W,Okazaki M,Lai J,Sugimoto S,Richardson SB,Kornfeld CG,Garbow JR,Patterson GA,Gelman AE,Kreisel D

    更新日期:2009-01-01 00:00:00

  • Preparing monodisperse macromolecular samples for successful biological small-angle X-ray and neutron-scattering experiments.

    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 ...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Jeffries CM,Graewert MA,Blanchet CE,Langley DB,Whitten AE,Svergun DI

    更新日期:2016-11-01 00:00:00

  • A protocol for phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood.

    abstract::Blood circulating endothelial cells (CECs) and circulating hematopoietic progenitor cells (CPCs) represent two cell populations that are thought to play important roles in tissue vascularization. CECs and CPCs are currently studied as surrogate markers in patients for more than a dozen pathologies, including heart dis...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Duda DG,Cohen KS,Scadden DT,Jain RK

    更新日期:2007-01-01 00:00:00

  • Publisher Correction: PEG-4MAL hydrogels for human organoid generation, culture, and in vivo delivery.

    abstract::In the version of this protocol originally published, the caption for Fig. 3 was erroneously placed with Fig. 4, and that for Fig. 4 was placed with Fig. 3. This error has been corrected in the HTML and PDF versions of the paper. ...

    journal_title:Nature protocols

    pub_type: 已发布勘误


    authors: Cruz-Acuña R,Quirós M,Huang S,Siuda D,Spence JR,Nusrat A,García AJ

    更新日期:2019-07-01 00:00:00

  • Integrated analysis of shotgun proteomic data with PatternLab for proteomics 4.0.

    abstract::PatternLab for proteomics is an integrated computational environment that unifies several previously published modules for the analysis of shotgun proteomic data. The contained modules allow for formatting of sequence databases, peptide spectrum matching, statistical filtering and data organization, extracting quantit...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Carvalho PC,Lima DB,Leprevost FV,Santos MD,Fischer JS,Aquino PF,Moresco JJ,Yates JR 3rd,Barbosa VC

    更新日期:2016-01-01 00:00:00

  • The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins.

    abstract::The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purification of corresponding Strep-tag II fusion protein...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Schmidt TG,Skerra A

    更新日期:2007-01-01 00:00:00

  • Cell-type-specific metabolic labeling, detection and identification of nascent proteomes in vivo.

    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


    authors: Alvarez-Castelao B,Schanzenbächer CT,Langer JD,Schuman EM

    更新日期:2019-02-01 00:00:00

  • Miniaturized parallel screens to identify chromatographic steps required for recombinant protein purification.

    abstract::Methods development in chromatography is a time-consuming, trial-and-error process that requires laborious experimentation. We describe a high-throughput screening (HTS) protocol for the rapid identification of chromatographic steps for protein purification from cell-free expression broths. Broths containing the prote...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Rege K,Heng M

    更新日期:2010-03-01 00:00:00

  • A surgical orthotopic organoid transplantation approach in mice to visualize and study colorectal cancer progression.

    abstract::Most currently available colorectal cancer (CRC) mouse models are not suitable for studying progression toward the metastatic stage. Recently, establishment of tumor organoid lines, either from murine CRC models or patients, and the possibility of engineering them with genome-editing technologies, have provided a larg...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Fumagalli A,Suijkerbuijk SJE,Begthel H,Beerling E,Oost KC,Snippert HJ,van Rheenen J,Drost J

    更新日期:2018-02-01 00:00:00

  • Microwave-assisted preparation of the quorum-sensing molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and structurally related analogs.

    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


    authors: Hodgkinson JT,Galloway WR,Welch M,Spring DR

    更新日期:2012-05-24 00:00:00

  • The touchscreen operant platform for assessing executive function in rats and mice.

    abstract::This protocol details a subset of assays developed within the touchscreen platform to measure various aspects of executive function in rodents. Three main procedures are included: extinction, measuring the rate and extent of curtailing a response that was previously, but is no longer, associated with reward; reversal ...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Mar AC,Horner AE,Nilsson SR,Alsiö J,Kent BA,Kim CH,Holmes A,Saksida LM,Bussey TJ

    更新日期:2013-10-01 00:00:00

  • Robust phosphoproteome enrichment using monodisperse microsphere-based immobilized titanium (IV) ion affinity chromatography.

    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


    authors: Zhou H,Ye M,Dong J,Corradini E,Cristobal A,Heck AJ,Zou H,Mohammed S

    更新日期:2013-03-01 00:00:00

  • Magnetic-based purification of untouched mouse germinal center B cells for ex vivo manipulation and biochemical analysis.

    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


    authors: Cato MH,Yau IW,Rickert RC

    更新日期:2011-06-09 00:00:00

  • Quantitative proteomic approach to study subcellular localization of membrane proteins.

    abstract::As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-stat...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Sadowski PG,Dunkley TP,Shadforth IP,Dupree P,Bessant C,Griffin JL,Lilley KS

    更新日期:2006-01-01 00:00:00

  • Genome-wide measurement of protein-DNA binding dynamics using competition ChIP.

    abstract::Competition chromatin immunoprecipitation (competition ChIP) enables experimenters to measure protein-DNA dynamics at a single locus or across the entire genome, depending on the detection method. Competition ChIP relies on a cell containing two copies of a single DNA-associated factor, with each copy of the factor di...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Lickwar CR,Mueller F,Lieb JD

    更新日期:2013-01-01 00:00:00