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https://www.readbyqxmd.com/read/29201946/cell-free-fluorescent-intra-golgi-retrograde-vesicle-trafficking-assay
#1
Nathanael P Cottam, Daniel Ungar
Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable cell lines were generated in which the trans-Golgi enzyme galactosyltransferase (GalT) was tagged with either CFP or YFP. Given that GalT is recycled to the cisterna where it is located at steady state, GalT-containing vesicles target GalT-containing cisternal membranes...
November 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29170752/proximal-ligation-assay-pla-on-lung-tissue-and-cultured-macrophages-to-demonstrate-protein-protein-interaction
#2
Roberto Mendez, Santanu Banerjee
In this protocol, we describe proximal ligation assay (PLA), an antibody-based detection method for protein-protein interaction. This method relies on specific binding of individual primary antibodies to the two putative interacting proteins. The primary antibodies need to have different hosts. The secondary antibodies against the two hosts have complementary oligonucleotide moieties attached to them. If the two antigens are in close proximity (presumably interacting with each other), the complementary oligonucleotides can anneal and fluorescent nucleotides can be incorporated in a single DNA polymerization step...
November 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29152538/isolation-of-primary-human-skeletal-muscle-cells
#3
Janelle M Spinazzola, Emanuela Gussoni
Primary myoblast culture is a valuable tool in research of muscle disease, pathophysiology, and pharmacology. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic population by fluorescence-activated cell sorting (FACS). We also describe methods for assessing myogenicity and population expansion for subsequent in vitro study.
November 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29170751/accurate-streamlined-analysis-of-mrna-translation-by-sucrose-gradient-fractionation
#4
Soufiane Aboulhouda, Rachael Di Santo, Gabriel Therizols, David Weinberg
The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to determine the number of ribosomes associated with an mRNA molecule, normalized to the length of the coding sequence. The primary method for this analysis of individual mRNAs is sucrose gradient fractionation, which physically separates mRNAs based on the number of bound ribosomes...
October 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29104897/protein-expression-and-purification-of-the-hsp90-cdc37-cdk4-kinase-complex-from-saccharomyces-cerevisiae
#5
Kliment A Verba, David A Agard
Interactions between Hsp90, its co-chaperone Cdc37 and kinases have been biochemically studied for over three decades and have been shown to be functionally important in organisms from yeast to humans. However, formation of a stable complex for structural studies has been elusive. In this protocol we describe expression and purification of Hsp90-Cdc37-Cdk4 kinase protein complex from Saccharomyces cerevisiae utilizing the viral 2A sequences to titrate the three proteins at similar levels.
October 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29082292/protocol-for-construction-of-a-tunable-crispr-interference-tcrispri-strain-for-escherichia-coli
#6
Xin-Tian Li, Cindy Sou, Suckjoon Jun
We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and nonessential genes. Most importantly from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step oligo recombineering. Additional advantages of tCRISPRi over other existing CRISPRi methods include: (1) tCRISPRi shows significantly less than 10% leaky repression; (2) tCRISPRi uses a tunable arabinose operon promoter and modifications in transporter genes to allow a wide dynamic range with graded control by arabinose inducer; (3) tCRISPRi is plasmid free and the entire system is integrated into the chromosome; (4) tCRISPRi strains show desirable physiological properties...
October 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29082291/dq-red-bsa-trafficking-assay-in-cultured-cells-to-assess-cargo-delivery-to-lysosomes
#7
Rituraj Marwaha, Mahak Sharma
Lysosomes are the terminal end of the endocytic pathway having acidic environment required for active hydrolases that degrade the cargo delivered to these compartments. This process of cargo delivery and degradation by endo-lysosomes is a tightly regulated process and important for maintaining cellular homeostasis. Cargos like EGF (Epidermal Growth Factor), Dil-LDL (3,3'-Dioctadecylindocarbocyanine-Low Density Lipoprotein), Dextran, DQ-BSA (Dye Quenched-Bovine Serum Albumin) etc., are routinely used by researchers to analyze the role of various proteins in endocytic pathway...
October 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29071286/caenorhabditis-elegans-microinjection
#8
Matthias Rieckher, Nektarios Tavernarakis
Microinjection is the most frequently used tool for genetic transformation of the nematode Caenorhabditis elegans, facilitating the transgenic expression of genes, genome editing by the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system, or transcription of dsRNA for RNA intereference (RNAi). Exogenous DNA is delivered into the developing oocytes in the germline of adult hermaphrodites, which then generate transgenic animals among their offspring. In this protocol, we describe the microinjection procedure and the subsequent selection of transgenic progeny...
October 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29130058/bioluminescence-monitoring-of-neuronal-activity-in-freely-moving-zebrafish-larvae
#9
Steven Knafo, Andrew Prendergast, Olivier Thouvenin, Sophie Nunes Figueiredo, Claire Wyart
The proof of concept for bioluminescence monitoring of neural activity in zebrafish with the genetically encoded calcium indicator GFP-aequorin has been previously described (Naumann et al., 2010) but challenges remain. First, bioluminescence signals originating from a single muscle fiber can constitute a major pitfall. Second, bioluminescence signals emanating from neurons only are very small. To improve signals while verifying specificity, we provide an optimized 4 steps protocol achieving: 1) selective expression of a zebrafish codon-optimized GFP-aequorin, 2) efficient soaking of larvae in GFP-aequorin substrate coelenterazine, 3) bioluminescence monitoring of neural activity from motor neurons in free-tailed moving animals performing acoustic escapes and 4) verification of the absence of muscle expression using immunohistochemistry...
September 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29130057/differentiation-of-myeloid-derived-suppressor-cells-from-murine-bone-marrow-and-their-co-culture-with-splenic-dendritic-cells
#10
Giada Mondanelli, Claudia Volpi
Myeloid-derived suppressor cells (MDSCs) possess the ability to suppress the immune response, and to amplify the regulatory properties of other immune cells, i.e., dendritic cells. Here we describe a protocol in which MDSCs were differentiated from murine bone marrow cells, and CD11c(+) dendritic cells were purified from murine spleens. MDSCs and CD11c dendritic cells can be co-cultured and the immunoregulatory phenotype of the MDSCs-conditioned dendritic cells could be assessed by means of a specific functional in vivo experiment, i...
September 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29082290/in-vitro-assays-for-eukaryotic-leading-lagging-strand-dna-replication
#11
Grant Schauer, Jeff Finkelstein, Mike O'Donnell
The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other proteins. We have previously established the mechanisms by which the polymerases ε and δ are targeted to their 'correct' strands, as well as quality control mechanisms that evict polymerases when they associate with an 'incorrect' strand...
September 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29082289/method-for-multiplexing-crispr-cas9-in-saccharomyces-cerevisiae-using-artificial-target-dna-sequences
#12
Rachael M Giersch, Gregory C Finnigan
Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded break. Eukaryotic repair systems allow for the introduction of exogenous DNA, repair of existing mutations, or deletion of endogenous gene products. Targeting of Cas9 to multiple genomic positions (termed 'multiplexing') is achieved by the expression of multiple sgRNAs within the same nucleus...
September 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29021994/stereotaxic-adeno-associated-virus-injection-and-cannula-implantation-in-the-dorsal-raphe-nucleus-of-mice
#13
Patrícia A Correia, Sara Matias, Zachary F Mainen
Optogenetic methods are now widespread in neuroscience research. Here we present a detailed surgical procedure to inject adeno-associated viruses and implant optic fiber cannulas in the dorsal raphe nucleus (DRN) of living mice. Combined with transgenic mouse lines, this protocol allows specific targeting of serotonin-producing neurons in the brain. It includes fixing a mouse in a stereotaxic frame, performing a craniotomy, virus injection and fiber implantation. Animals can be later used in behavioral experiments, combined with optogenetic manipulations (Dugué et al...
September 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29094061/fm1-43-photoconversion-and-electron-microscopy-analysis-at-the-drosophila-neuromuscular-junction
#14
Nadezhda S Sabeva, Maria Bykhovskaia
We developed a protocol for photoconversion of endocytic marker FM1-43 followed by electron microscopy analysis of synaptic boutons at the Drosophila neuromuscular junction. This protocol allows detection of stained synaptic vesicle even when release rates are very low, such as during the spontaneous release mode. The preparations are loaded with the FM1-43 dye, pre-fixed, treated and illuminated to photoconvert the dye, and then processed for conventional electron microscopy. This procedure enables clear identification of stained synaptic vesicles at electron micrographs...
September 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29075656/rearing-of-culex-spp-and-aedes-spp-mosquitoes
#15
Elizabeth Kauffman, Anne Payne, Mary A Franke, Michael A Schmid, Eva Harris, Laura D Kramer
Mosquito-transmitted pathogens cause major public health problems and contribute substantially to the global burden of disease. Aedes mosquitoes transmit dengue, Zika, yellow fever, and Chikungunya viruses; Culex mosquitoes transmit West Nile, Japanese encephalitis, and Saint Louis encephalitis viruses, among others. Experiments utilizing laboratory-reared colonized mosquitoes can address many issues such as vector biology, vector competence, vector-pathogen interaction, and vector control. The establishment of healthy and standardized mosquito colonies requires generation and implementation of protocols, attention to detail, and an understanding of the factors that affect mosquito fitness, such as temperature and humidity, nutrient quality and availability, population density, blood feeding and mating behavior, and egg-laying requirements...
September 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29057295/multicolor-stimulated-emission-depletion-sted-microscopy-to-generate-high-resolution-images-of-respiratory-syncytial-virus-particles-and-infected-cells
#16
Masfique Mehedi, Margery Smelkinson, Juraj Kabat, Sundar Ganesan, Peter L Collins, Ursula J Buchholz
Human respiratory syncytial virus (RSV) infection in human lung epithelial A549 cells induces filopodia, cellular protrusions consisting of F-actin, that extend to neighboring uninfected cells (Mehedi et al., 2016). High-resolution imaging via stimulated emission depletion (STED) microscopy revealed filamentous RSV particles along these filopodia, suggesting that filopodia facilitate RSV cell-to-cell spread (Mehedi et al., 2016). In this protocol, we describe how to fix, permeabilize, immunostain, and mount RSV-infected A549 cells for STED imaging...
September 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28979923/isolation-of-mouse-cardiac-neural-crest-cells-and-their-differentiation-into-smooth-muscle-cells
#17
Xia Wang, Sophie Astrof
Cardiac neural crest cells (CNCCs) originate at the dorsal edge of the neural tube between the otic pit and the caudal edge of the 3(rd) somite, and migrate into the pharyngeal arches and the heart. We have shown that fibronectin (Fn1) plays an important role in the development of the CNCC by regulating the differentiation of CNCCs into vascular smooth muscle cells around pharyngeal arch arteries (Wang and Astrof, 2016). This protocol describes the isolation of CNCCs from the neural tube and from the caudal pharyngeal arches, and the differentiation of neural crest-derived cells into smooth muscle cells...
September 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28966949/protocol-for-establishing-a-multiplex-image-based-autophagy-rnai-screen-in-cell-cultures
#18
Jennifer Jung, Christian Behrends
Autophagy is a recycling pathway, in which intracellular cargoes including protein aggregates and bacteria are engulfed by autophagosomes and subsequently degraded after fusion with lysosomes. Dysregulation of this process is involved in several human diseases such as cancer or neurodegeneration. Hence, advancing our understanding of how autophagy is regulated provides an opportunity to explore druggable targets and subsequently develop treatment strategies for these diseases. To identify novel autophagy regulators, we developed an image-based phenotypic RNAi screening approach using autophagic marker proteins at endogenous levels (Jung et al...
September 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28966948/labelling-halotag-fusion-proteins-with-halotag-ligand-in-living-cells
#19
Huy Nguyen Duc, Xiaojun Ren
HaloTag has been widely used to label proteins in vitro and in vivo (Los et al., 2008). In this protocol, we describe labelling HaloTag-Cbx fusion proteins by HaloTag ligands for live-cell single-molecule imaging (Zhen et al., 2016).
September 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28966947/purifying-properly-folded-cysteine-rich-zinc-finger-containing-recombinant-proteins-for-structural-drug-targeting-studies-the-ch1-domain-of-p300-as-a-case-example
#20
Yong Joon Kim, Stefan Kaluz, Anil Mehta, Emily Weinert, Shannon Rivera, Erwin G Van Meir
The transcription factor Hypoxia-Inducible Factor (HIF) complexes with the coactivator p300, activating the hypoxia response pathway and allowing tumors to grow. The CH1 and CAD domains of each respective protein form the interface between p300 and HIF. Small molecule compounds are in development that target and inhibit HIF/p300 complex formation, with the goal of reducing tumor growth. High resolution NMR spectroscopy is necessary to study ligand interaction with p300-CH1, and purifying high quantities of properly folded p300-CH1 is needed for pursuing structural and biophysical studies...
September 5, 2017: Bio-protocol
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