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Biochemistry

Heparan Sulfate Identification and Characterisation: Method I. Heparan Sulfate Identification by NMR Analysis Authors:  Susan M. Carnachan and Simon F.R. Hinkley, date: 04/05/2017, view: 2288, Q&A: 0
Heparin and heparan sulfate (HS) may be purified from complex biological matrices and are often isolated in sub-milligram quantities but not unequivocally identified by spectroscopic means. This protocol details a methodology to rapidly assess the gross compositional features and approximate purity of HS by 1H nuclear magnetic ...
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Heparan Sulfate Identification and Characterisation: Method II. Enzymatic Depolymerisation and SAX-HPLC Analysis to Determine Disaccharide Composition Authors:  Susan M. Carnachan and Simon F.R. Hinkley, date: 04/05/2017, view: 2427, Q&A: 0
Heparan sulfate (HS) is purified from complex matrices and often not fully characterised to validate its assignment. The characterisation of heparins and heparan sulfates through enzymatic depolymerisation and subsequent strong anion-exchange high performance liquid chromatography (SAX-HPLC) analysis and quantitation of the resulting disaccharides ...
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Isolation and Analysis of Proteoglycans and Glycosaminoglycans from Archaeological Bones and Teeth Authors:  Yvette May Coulson-Thomas, Vivien Jane Coulson-Thomas and Andrew L. Norton, date: 04/05/2017, view: 2000, Q&A: 0
We have developed methods for isolating proteoglycans and glycosaminoglycans from archaeological bones and teeth. These methods have been previously published (Coulson-Thomas et al., 2015) and are described here in more detail. In the case of glycosaminoglycans, the method was a previously described method (Nader et al., 1999) ...
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Secretion of Adipsin as an Assay to Measure Flux from the Endoplasmic Reticulum (ER) Authors:  Alexandria Brumfield, Natasha Chaudhary and Timothy E. McGraw, date: 04/05/2017, view: 2178, Q&A: 0
In this protocol we describe a quantitative biochemical assay to assess the efficiency of endoplasmic reticulum (ER) to Golgi protein transport in adipocytes (Bruno et al., 2016). The assay takes advantage of the fact that adipocytes secrete various bioactive proteins, known as adipokines. As a measure of ER to Golgi flux we determine the ...
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Nitroxide Labeling of Proteins and the Determination of Paramagnetic Relaxation Derived Distance Restraints for NMR Studies Authors:  Megan Sjodt and Robert T. Clubb, date: 04/05/2017, view: 3079, Q&A: 0
Site-specific attachment of paramagnetic spin labels to biomolecules causes distance-dependent line-broadening effects, which can be exploited to study the structure and dynamics of these molecules in solution. This protocol describes how to attach nitroxide spin labels to proteins and how to collect and analyze NMR data using these labeled ...
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Cell Biology

In vivo Live Imaging of Calcium Waves and Other Cellular Processes during Fertilization in Caenorhabditis elegans Authors:  Jun Takayama, Masashi Fujita and Shuichi Onami, date: 04/05/2017, view: 3113, Q&A: 0
Fertilization calcium waves are a conserved trigger for animal development; however, genetic analysis of these waves has been limited due to the difficulty of imaging in vivo fertilization. Here we describe a protocol to image calcium dynamics during in vivo fertilization in the genetic animal model Caenorhabditis elegans ...
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Gliding Assay to Analyze Microtubule-based Motor Protein Dynamics Authors:  Albert Shim, Gin Tezuka, Luke Kupcha and Li Tao, date: 04/05/2017, view: 3347, Q&A: 0
The purpose of this protocol is to provide an updated method of performing microtubule gliding assays and visualizing it using fluorescence microscopy.
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In vitro Microtubule Bundling Assay under Physiological Conditions Authors:  Zach Geisterfer, Gin Tezuka and Li Tao, date: 04/05/2017, view: 2393, Q&A: 0
Kinesins play a role in organizing the mitotic spindle through the crosslinking of microtubules (MTs), made possible through binding sites at opposite ends of the holoenzyme. Here, we developed a method to test kinesin MT crosslinking action under physiological conditions.
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Isolation of Mononuclear Cell Populations from Ovarian Carcinoma Ascites Authors:  Christina Wefers, Ghaith Bakdash, Meritxell Moreno Martin, Tjitske Duiveman-de Boer, Ruurd Torensma, Leon F.A.G. Massuger and I. Jolanda M. de Vries, date: 04/05/2017, view: 2781, Q&A: 0
Ovarian cancer is one of the most fatal tumors in women. Due to a lack of symptoms and adequate screening methods, patients are diagnosed at advanced stages with extensive tumor burden (Jelovac and Armstrong, 2011). Interestingly, ovarian cancer metastasis is generally found within the peritoneal cavity rather than other tissues (Lengyel, 2010; ...
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Developmental Biology

In vivo Mitophagy Monitoring in Caenorhabditis elegans to Determine Mitochondrial Homeostasis Authors:  Konstantinos Palikaras and Nektarios Tavernarakis, date: 04/05/2017, view: 2701, Q&A: 0
Perturbation of mitochondrial function is a major hallmark of several pathological conditions and ageing, underlining the essential role of fine-tuned mitochondrial activity (Lopez-Otin et al., 2013). Mitochondrial selective autophagy, known as mitophagy, mediates the removal of dysfunctional and/or superfluous organelles, preserving ...
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Assessment of Murine Retinal Function by Electroretinography Authors:  Gillie Benchorin, Melissa A. Calton, Marielle O. Beaulieu and Douglas Vollrath, date: 04/05/2017, view: 4169, Q&A: 0
The electroretinogram (ERG) is a sensitive and noninvasive method for testing retinal function. In this protocol, we describe a method for performing ERGs in mice. Contact lenses on the mouse cornea measure the electrical response to a light stimulus of photoreceptors and downstream retinal cells, and the collected data are analyzed to evaluate ...
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Immunology

In vitro Detection of Neutrophil Traps and Post-attack Cell Wall Changes in Candida Hyphae Authors:  Alex Hopke and Robert T. Wheeler, date: 04/05/2017, view: 2572, Q&A: 0
In this protocol we describe how to visualize neutrophil extracellular traps (NETs) and fungal cell wall changes in the context of the coculture of mouse neutrophils with fungal hyphae of Candida albicans. These protocols are easily adjusted to test a wide array of hypotheses related to the impact of immune cells on fungi and the cell ...
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Isolation of Exosomes from Semen for in vitro Uptake and HIV-1 Infection Assays Authors:  Marisa N. Madison, Jennifer L. Welch and Chioma M. Okeoma, date: 04/05/2017, view: 2929, Q&A: 0
Exosomes are membranous extracellular nanovesicles of endocytic origin. Exosomes are known to carry host and pathogen-derived genomic, proteomic, lipidomic cargos and other extraneous molecules. Exosomes are secreted by diverse cell types into the extracellular milieu and are subsequently internalized by recipient neighboring or distal cells. Upon ...
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Screening for Novel Endogenous Inflammatory Stimuli Using the Secreted Embryonic Alkaline Phosphatase NF-κB Reporter Assay Authors:  Lorena Zuliani-Alvarez, Anna M. Piccinini and Kim S Midwood, date: 04/05/2017, view: 2754, Q&A: 0
An immune response can be activated by pathogenic stimuli, as well as endogenous danger signals, triggering the activation of pattern recognition receptors and initiating signalling cascades that lead to inflammation. This method uses THP1-BlueTM cells, a human monocytic cell line which contains an embryonic alkaline phosphatase ...
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Microbiology

RNA Strand Displacement Assay for Hepatitis E Virus Helicase Authors:  Vidya P. Nair and Milan Surjit, date: 04/05/2017, view: 2453, Q&A: 0
The hepatitis E virus (HEV) helicase uses ATP to unwind the RNA duplexes. This is an essential step for viral replication. This protocol aims to measure the double strand RNA unwinding activity of the HEV helicase.
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RNA-dependent RNA Polymerase Assay for Hepatitis E Virus Authors:  Vidya P. Nair, Saumya Anang, Akriti Srivastava and Milan Surjit, date: 04/05/2017, view: 2812, Q&A: 0
RNA-dependent RNA polymerase (RdRp) is essential for the replication of viral RNA for RNA viruses. It synthesizes the complementary strand of viral genomic RNA, which is used subsequently as a template to generate more copies of viral genome. This assay measures activity of the hepatitis E virus (HEV) RdRp. In contrast to protocols available to ...
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Measurement of the Galactanase Activity of the GanB Galactanase Protein from Bacillus subtilis Author:  Hildegard Watzlawick, date: 04/05/2017, view: 2222, Q&A: 0
The activity of the endo-β-1,4-galactanase GanB from B. subtilis on the high molecular weight β-1,4-galactan was determined quantitatively by the measurement of the increase of the reducing power or with the dyed substrate Azo-galactan. The generated degradation products were analyzed using thinlayer-chromatography (TLC) or ...
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ARP2/3 Phosphorylation Assay in the Presence of Recombinant Bacterial Effectors Authors:  Céline Michard, Lawrence L. LeClaire and Patricia Doublet, date: 04/05/2017, view: 2228, Q&A: 0
The Actin-Related Protein 2/3 (ARP2/3) complex is an actin nucleator that generates a branched actin network in mammalian cells. In addition to binding nucleation promoting factors, LeClaire et al. demonstrated that its phosphorylation state is essential key for its activity (LeClaire et al., 2008). In cells, the ARP2/3 complex ...
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Purification of N-coronafacoyl Phytotoxins from Streptomyces scabies Authors:  Luke Bown and Dawn R. D. Bignell, date: 04/05/2017, view: 2107, Q&A: 0
This procedure is used for large-scale purification of N-coronafacoyl phytotoxins that are produced by the potato common scab pathogen Streptomyces scabies. The procedure employs organic extraction of S. scabies culture supernatants under alternating basic and acidic conditions in order to preferentially isolate the ...
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Molecular Biology

Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery Authors:  Giel Vandemoortele, Delphine De Sutter and Sven Eyckerman, date: 04/05/2017, view: 5082, Q&A: 0
The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair ...
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Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9 Authors:  Lena Behrmann, Scott McComb, Júlia Aguadé-Gorgorió, Yun Huang, Mario Hermann, Pawel Pelczar, Adriano Aguzzi, Jean-Pierre Bourquin and Beat C. Bornhauser, date: 04/05/2017, view: 5581, Q&A: 0
CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously ...
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Neuroscience

Measuring Behavioral Individuality in the Acoustic Startle Behavior in Zebrafish Authors:  Carlos Pantoja, Adam Hoagland, Elizabeth Carroll, David Schoppik and Ehud Y. Isacoff, date: 04/05/2017, view: 2714, Q&A: 0
The objective of this protocol is to provide a detailed description for the construction and use of a behavioral apparatus, the zBox, for high-throughput behavioral measurements in larval zebrafish (Danio rerio). The zBox is used to measure behavior in multiple individuals simultaneously. Individual fish are housed in wells of multi-well ...
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Testing Depression in Mice: a Chronic Social Defeat Stress Model Authors:  Hee-Dae Kim, Tanessa Call, Samantha Carotenuto, Ross Johnson and Deveroux Ferguson, date: 04/05/2017, view: 4636, Q&A: 0
A vast challenge within neuropsychiatric research has been the development of animal models that accurately reflect symptoms associated with affective disorders. An ethologically valid model that has been shown to be effective in studying depression is the chronic social defeat stress model. In this model, C57BL/6J mice are subjected to chronic ...
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Plant Science

Resin-embedded Thin-section Immunohistochemistry Coupled with Triple Cellular Counterstaining Authors:  William M Palmer, John W Patrick and Yong-Ling Ruan, date: 04/05/2017, view: 1925, Q&A: 0
This protocol was developed to study protein localisation within the vascular bundles of developing tomato fruit however, it can be applied to any resin embedded plant tissue. The vascular bundle is comprised of many different cells that all have unique properties. The mature sieve elements are enucleated and contain sieve plates that comprise of ...
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Analyses of Root-secreted Acid Phosphatase Activity in Arabidopsis Authors:  Liangsheng Wang and Dong Liu, date: 04/05/2017, view: 2799, Q&A: 0
Induction and secretion of acid phosphatase (APase) is a universal adaptive response of higher plants to low-phosphate stress (Tran et al., 2010). The intracellular APases are likely involved in the remobilization and recycling of phosphate (Pi) from intracellular Pi reserves, whereas the extracellular or secreted APases are believed to ...
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Use of Geminivirus for Delivery of CRISPR/Cas9 Components to Tobacco by Agro-infiltration Authors:  Kangquan Yin, Ting Han and Yule Liu, date: 04/05/2017, view: 3914, Q&A: 0
CRISPR/Cas9 system is a recently developed genome editing tool, and its power has been demonstrated in many organisms, including some plant species (Wang et al., 2016). In eukaryotes, the Cas9/gRNA complexes target genome sites specifically and cleave them to produce double-strand breaks (DSBs), which can be repaired by non-homologous end ...
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Stem Cell

A Co-culture Model for Determining the Target Specificity of the de novo Generated Retinal Ganglion Cells Authors:  Pooja Teotia, Matthew J. Van Hook and Iqbal Ahmad, date: 04/05/2017, view: 2435, Q&A: 0
In glaucoma, the output neurons of the retina, the retinal ganglion cells (RGCs), progressively degenerate, leading to irreversible blindness (Ahram et al., 2015). The ex vivo stem cell method to replace degenerated RGCs remains a potentially viable approach (Levin et al., 2004). However, the success of the approach ...
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