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Nature Protocols

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https://www.readbyqxmd.com/read/28079880/nonradioactive-quantification-of-autophagic-protein-degradation-with-l-azidohomoalanine-labeling
#1
Jigang Wang, Jianbin Zhang, Yew Mun Lee, Shukie Ng, Yin Shi, Zi-Chun Hua, Qingsong Lin, Han-Ming Shen
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...
December 2017: Nature Protocols
https://www.readbyqxmd.com/read/28796235/development-of-fertile-mouse-oocytes-from-mitotic-germ-cells-in-vitro
#2
Kanako Morohaku, Yuji Hirao, Yayoi Obata
Mammalian fetal ovaries contain numerous primordial germ cells (PGCs), although few mature oocytes are obtained from females, owing to apoptosis and follicle atresia. The regulatory mechanisms underlying oogenesis/folliculogenesis remain unknown. Development of methods for obtaining mature oocytes from PGCs in fetal ovaries in vitro could contribute to clarifying these mechanisms. The failure of follicle assembly has been found to be the most challenging aspect in conventional culture conditions. Recently, we established novel culture conditions that enable successful follicle assembly, sustaining interactions between the oocyte and somatic cells, and, in turn, promoting oocyte growth and maturation...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28796234/electron-microscopy-using-the-genetically-encoded-apex2-tag-in-cultured-mammalian-cells
#3
Jeffrey D Martell, Thomas J Deerinck, Stephanie S Lam, Mark H Ellisman, Alice Y Ting
Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28796233/intracellular-directed-evolution-of-proteins-from-combinatorial-libraries-based-on-conditional-phage-replication
#4
Andreas K Brödel, Alfonso Jaramillo, Mark Isalan
Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. These include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28796232/reconstitution-of-mouse-oogenesis-in-a-dish-from-pluripotent-stem-cells
#5
Katsuhiko Hayashi, Orie Hikabe, Yayoi Obata, Yuji Hirao
This protocol is an extension to: Nat. Protoc. 8, 1513-1524 (2013); doi: 10.1038/nprot.2013.090; published online 11 July 2013Generation of functional oocytes in culture from pluripotent stem cells should provide a useful model system for improving our understanding of the basic mechanisms underlying oogenesis. In addition, it has potential applications as an alternative source of oocytes for reproduction. Using the most advanced mouse model in regard to reproductive engineering and stem cell biology, we previously developed a culture method that produces functional primorial germ cells starting from pluripotent cells in culture and described it in a previous protocol...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28771239/use-of-fluorescence-detected-sedimentation-velocity-to-study-high-affinity-protein-interactions
#6
Sumit K Chaturvedi, Jia Ma, Huaying Zhao, Peter Schuck
Sedimentation velocity (SV) analytical ultracentrifugation (AUC) is a classic technique for the real-time observation of free macromolecular migration in solution driven by centrifugal force. This enables the analysis of macromolecular mass, shape, size distribution, and interactions. Although traditionally limited to determination of the sedimentation coefficient and binding affinity of proteins in the micromolar range, the implementation of modern detection and data analysis techniques has resulted in marked improvements in detection sensitivity and size resolution during the past decades...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28771238/the-mini-flotac-technique-for-the-diagnosis-of-helminth-and-protozoan-infections-in-humans-and-animals
#7
Giuseppe Cringoli, Maria P Maurelli, Bruno Levecke, Antonio Bosco, Jozef Vercruysse, Jürg Utzinger, Laura Rinaldi
This protocol is an extension to: Nat. Protoc. 5, 503-515 (2010); doi: 10.1038/nprot.2009.235; published online 25 February 2010The FLOTAC is a sensitive, accurate, and precise technique for the diagnosis of protozoan and helminth infections in humans and animals. However, it requires centrifugation, and hence might be out of reach in resource-constrained settings. As an extension of the original FLOTAC protocol, this protocol describes the Mini-FLOTAC technique, a logical evolution of FLOTAC conceived to perform multivalent, qualitative, and quantitative diagnosis of helminth and protozoan infections in human and animal feces, and urine...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28771237/mechanical-isolation-and-measurement-of-force-and-myoplasmic-free-ca-2-in-fully-intact-single-skeletal-muscle-fibers
#8
Arthur J Cheng, Håkan Westerblad
Mechanical dissection of single intact mammalian skeletal muscle fibers permits real-time measurement of intracellular properties and contractile function of living fibers. A major advantage of mechanical over enzymatic fiber dissociation is that single fibers can be isolated with their tendons remaining attached, which allows contractile forces (in the normal expected range of 300-450 kN/m(2)) to be measured during electrical stimulation. Furthermore, the sarcolemma of single fibers remains fully intact after mechanical dissection, and hence the living fibers can be studied with intact intracellular milieu and normal function and metabolic properties, as well as ionic control...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28771236/the-cubicon-method-for-concentrating-membrane-proteins-in-the-cubic-mesophase
#9
Pikyee Ma, Dietmar Weichert, Luba A Aleksandrov, Timothy J Jensen, John R Riordan, Xiangyu Liu, Brian K Kobilka, Martin Caffrey
The lipid cubic phase (in meso) method is an important approach for generating crystals and high-resolution X-ray structures of integral membrane proteins. However, as a consequence of instability, it can be impossible-using traditional methods-to concentrate certain membrane proteins and complexes to values suitable for in meso crystallization and structure determination. The cubicon method described here exploits the amphiphilic nature of membrane proteins and their natural tendency to partition preferentially into lipid bilayers from aqueous solution...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28749931/quantitative-multiplexed-workflow-for-deep-analysis-of-human-blood-plasma-and-biomarker-discovery-by-mass-spectrometry
#10
Hasmik Keshishian, Michael W Burgess, Harrison Specht, Luke Wallace, Karl R Clauser, Michael A Gillette, Steven A Carr
Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28749930/nontargeted-virus-sequence-discovery-pipeline-and-virus-clustering-for-metagenomic-data
#11
David Paez-Espino, Georgios A Pavlopoulos, Natalia N Ivanova, Nikos C Kyrpides
The analysis of large microbiome data sets holds great promise for the delineation of the biological and metabolic functioning of living organisms and their role in the environment. In the midst of this genomic puzzle, viruses, especially those that infect microbial communities, represent a major reservoir of genetic diversity with great impact on biogeochemical cycles and organismal health. Overcoming the limitations associated with virus detection directly from microbiomes can provide key insights into how ecosystem dynamics are modulated...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28749929/chemoenzymatic-synthesis-of-glycoengineered-igg-antibodies-and-glycosite-specific-antibody-drug-conjugates
#12
Feng Tang, Lai-Xi Wang, Wei Huang
Glycoengineered therapeutic antibodies and glycosite-specific antibody-drug conjugates (gsADCs) have generated great interest among researchers because of their therapeutic potential. Endoglycosidase-catalyzed in vitro glycoengineering technology is a powerful tool for IgG Fc (fragment cystallizable) N-glycosylation remodeling. In this protocol, native heterogeneously glycosylated IgG N-glycans are first deglycosylated with a wild-type endoglycosidase. Next, a homogeneous N-glycan substrate, presynthesized as described here, is attached to the remaining N-acetylglucosamine (GlcNAc) of IgG, using a mutant endoglycosidase (also called endoglycosynthase) that lacks hydrolytic activity but possesses transglycosylation activity for glycoengineering...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28726849/o2-controllable-hydrogels-for-studying-cellular-responses-to-hypoxic-gradients-in-three-dimensions-in-vitro-and-in-vivo
#13
Daniel M Lewis, Michael R Blatchley, Kyung Min Park, Sharon Gerecht
Oxygen (O2) acts as a potent upstream regulator of cell function. In both physiological and pathophysiological microenvironments, the O2 concentration is not uniformly distributed but instead follows a gradient that depends on distance from oxygen-carrying blood vessels. Such gradients have a particularly important role in development, tissue regeneration, and tumor growth. In this protocol, we describe how to use our previously reported gelatin-based O2-controllable hydrogels that can provide hypoxic microenvironments in vitro...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28726848/patch-clamp-technique-to-characterize-ion-channels-in-enlarged-individual-endolysosomes
#14
Cheng-Chang Chen, Chunlei Cang, Stefanie Fenske, Elisabeth Butz, Yu-Kai Chao, Martin Biel, Dejian Ren, Christian Wahl-Schott, Christian Grimm
According to proteomics analyses, more than 70 different ion channels and transporters are harbored in membranes of intracellular compartments such as endosomes and lysosomes. Malfunctioning of these channels has been implicated in human diseases such as lysosomal storage disorders, neurodegenerative diseases and metabolic pathologies, as well as in the progression of certain infectious diseases. As a consequence, these channels have engendered very high interest as future drug targets. Detailed electrophysiological characterization of intracellular ion channels is lacking, mainly because standard methods to analyze plasma membrane ion channels, such as the patch-clamp technique, are not readily applicable to intracellular organelles...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28726847/mapping-genome-wide-transcription-factor-binding-sites-using-dap-seq
#15
Anna Bartlett, Ronan C O'Malley, Shao-Shan Carol Huang, Mary Galli, Joseph R Nery, Andrea Gallavotti, Joseph R Ecker
To enable low-cost, high-throughput generation of cistrome and epicistrome maps for any organism, we developed DNA affinity purification sequencing (DAP-seq), a transcription factor (TF)-binding site (TFBS) discovery assay that couples affinity-purified TFs with next-generation sequencing of a genomic DNA library. The method is fast, inexpensive, and more easily scaled than chromatin immunoprecipitation sequencing (ChIP-seq). DNA libraries are constructed using native genomic DNA from any source of interest, preserving cell- and tissue-specific chemical modifications that are known to affect TF binding (such as DNA methylation) and providing increased specificity as compared with in silico predictions based on motifs from methods such as protein-binding microarrays (PBMs) and systematic evolution of ligands by exponential enrichment (SELEX)...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28703790/a-fluorescence-based-imaging-method-to-measure-in-vitro-and-in-vivo-mitophagy-using-mt-keima
#16
Nuo Sun, Daniela Malide, Jie Liu, Ilsa I Rovira, Christian A Combs, Toren Finkel
Mitophagy is a cellular process that selectively removes damaged, old or dysfunctional mitochondria. Defective mitophagy is thought to contribute to normal aging and to various neurodegenerative and cardiovascular diseases. Previous methods used to detect mitophagy in vivo were cumbersome, insensitive and difficult to quantify. We created a transgenic mouse model that expresses the pH-dependent fluorescent protein mt-Keima in order to more readily assess mitophagy. Keima is a pH-sensitive, dual-excitation ratiometric fluorescent protein that also exhibits resistance to lysosomal proteases...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28703789/capturing-suboptical-dynamic-structures-in-lipid-bilayer-patches-formed-from-free-standing-giant-unilamellar-vesicles
#17
Tripta Bhatia, Flemming Cornelius, John H Ipsen
There is accumulating evidence that the small-scale lateral organization of biological membranes has a crucial role in signaling and trafficking in cells. However, it has been difficult to characterize these features with existing methods for preparing and analyzing freestanding membranes, because the dynamics occurs below the optical resolution possible with these protocols. We have developed a protocol that permits the imaging of lipid nanodomains and lateral protein organization in membranes of giant unilamellar vesicles (GUVs)...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28703788/preparation-of-a-trp-bodipy-fluorogenic-amino-acid-to-label-peptides-for-enhanced-live-cell-fluorescence-imaging
#18
Lorena Mendive-Tapia, Ramon Subiros-Funosas, Can Zhao, Fernando Albericio, Nick D Read, Rodolfo Lavilla, Marc Vendrell
Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C2-BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging-an approach that is applicable to most peptide sequences...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28683064/murine-chronic-lymph-node-window-for-longitudinal-intravital-lymph-node-imaging
#19
Eelco F J Meijer, Han-Sin Jeong, Ethel R Pereira, Thomas A Ruggieri, Cedric Blatter, Benjamin J Vakoc, Timothy P Padera
Chronic imaging windows in mice have been developed to allow intravital microscopy of many different organs and have proven to be of paramount importance in advancing our knowledge of normal and disease processes. A model system that allows long-term intravital imaging of lymph nodes would facilitate the study of cell behavior in lymph nodes during the generation of immune responses in a variety of disease settings and during the formation of metastatic lesions in cancer-bearing mice. We describe a chronic lymph node window (CLNW) surgical preparation that allows intravital imaging of the inguinal lymph node in mice...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28683063/shear-thinning-and-self-healing-hydrogels-as-injectable-therapeutics-and-for-3d-printing
#20
Claudia Loebel, Christopher B Rodell, Minna H Chen, Jason A Burdick
The design of injectable hydrogel systems addresses the growing demand for minimally invasive approaches for local and sustained delivery of therapeutics. We developed a class of hyaluronic acid (HA) hydrogels that form through noncovalent guest-host interactions, undergo disassembly (shear-thinning) when injected through a syringe and then reassemble within seconds (self-healing) when shear forces are removed. Its unique properties enable the use of this hydrogel system for numerous applications, such as injection in vivo (including with cells and therapeutic molecules) or as a 'bioink' in 3D-printing applications...
August 2017: Nature Protocols
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