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Past Issues in 2017

Volume: 7 Issue: 15

Biochemistry

Membrane Lipid Screen to Identify Molecular Targets of Biomolecules Authors: John R. Jimah, Paul H. Schlesinger and Niraj H. Tolia, date: 08/05/2017, view: 6010, Q&A: 0

Proteins that bind to and disrupt cell membranes may target specific phospholipids. Here we describe a protocol to identify the lipid targets of proteins and biomolecules. First, we describe a screen to identify lipids in membranes that are specifically bound by the biomolecule of interest. Second, we describe a method for determining if the ...

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Purification of FLAG-tagged Secreted Proteins from Mammalian Cells Authors: Eisuke Itakura, Changchun Chen and Mario de Bono, date: 08/05/2017, view: 7144, Q&A: 0

This protocol describes a method for purifying glycosylated FLAG-tagged secreted proteins with disulfide bonds from mammalian cells. The purified products can be used for various applications, such as ligand binding assays.

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Separation and Purification of Glycosaminoglycans (GAGs) from Caenorhabditis elegans Authors: Tabea Dierker and Lena Kjellén, date: 08/05/2017, view: 7953, Q&A: 1

The nematode Caenorhabditis elegans is a popular model organism for studies of developmental biology, neurology, ageing and other fields of basic research. Because many developmental processes are regulated by glycosaminoglyans (GAGs) on cell surfaces and in the extracellular matrix, methods to isolate and analyze C. elegans GAGs ...

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Isolation of Keratan Sulfate Disaccharide-branched Chondroitin Sulfate E from Mactra chinensis Authors: Kyohei Higashi and Toshihiko Toida, date: 08/05/2017, view: 4886, Q&A: 0

Glycosaminoglycans (GAGs) including chondroitin sulfate (CS), dermatan sulfate (DS), heparin (HP), heparan sulfate (HS) and keratan sulfate (KS) are linear, sulfated repeating disaccharide sequences containing hexosamine and uronic acid (or galactose in the case of KS). Recently, a keratan sulfate (KS) disaccharide [GlcNAc6S ...

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Cancer Biology

RNA Interference Screening to Identify Proliferation Determinants in Breast Cancer Cells Authors: Yong-Wei Zhang, Rochelle E. Nasto, Sandra A. Jablonski, Ilya G Serebriiskii, Rishi Surana, Joseph Murray, Michael Johnson, Rebecca B. Riggins, Robert Clarke, Erica A. Golemis and Louis M. Weiner, date: 08/05/2017, view: 5658, Q&A: 0

RNAi screening technology has revealed unknown determinants of various biological signaling pathways in biomedical studies. This protocol provided detailed information about how to use RNAi screening to identify proliferation determinants in breast tumor cells. siRNA-based libraries targeting against Estrogen receptor (ER)-network, including 631 ...

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Microbiology

Analysis of N-acetylmuramic acid-6-phosphate (MurNAc-6P) Accumulation by HPLC-MS Authors: Marina Borisova and Christoph Mayer, date: 08/05/2017, view: 5697, Q&A: 0

We describe here in detail a high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based method to determine N-acetylmuramic acid-6-phosphate (MurNAc-6P) in bacterial cell extracts. The method can be applied to both Gram-negative and Gram-positive bacteria, and as an example we use Escherichia coli cells in this ...

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Advanced Design of Minimalistic Dumbbell-shaped Gene Expression Vectors Authors: Xiaoou Jiang and Volker Patzel, date: 08/05/2017, view: 6358, Q&A: 0

Minimal DNA vectors exclusively comprising therapeutically relevant sequences hold great promise for the development of novel therapeutic regimen. Dumbbell-shaped vectors represent non-viral non-integrating DNA minimal vectors which have entered an advanced stage of clinical development (Hardee et al., 2017). Spliceable introns and DNA ...

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Measurement of Energy-dependent Rhodamine 6G Efflux in Yeast Species Authors: Yvetta Gbelska, Nora Toth Hervay, Vladimira Dzugasova and Alexandra Konecna, date: 08/05/2017, view: 5813, Q&A: 0

Rhodamine 6G is a highly fluorescent dye often used to determine the transport activity of yeast membrane efflux pumps. The ATP-binding cassette transporter KlPdr5p confers resistance to several unrelated drugs in Kluyveromyces lactis. KlPdr5p also extrudes rhodamine 6G (R6G) from intact yeast cells in an ...

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Selection of Genetically Modified Bacteriophages Using the CRISPR-Cas System Authors: Miriam Manor and Udi Qimron, date: 08/05/2017, view: 7316, Q&A: 0

We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous ...

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Liposome Disruption Assay to Examine Lytic Properties of Biomolecules Authors: John R. Jimah, Paul H. Schlesinger and Niraj H. Tolia, date: 08/05/2017, view: 8702, Q&A: 0

Proteins may have three dimensional structural or amino acid features that suggest a role in targeting and disrupting lipids within cell membranes. It is often necessary to experimentally investigate if these proteins and biomolecules are able to disrupt membranes in order to conclusively characterize the function of these biomolecules. Here, we ...

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Observation of Pneumococcal Phase Variation in Colony Morphology Authors: Jing Li, Juanjuan Wang, Fangfang Jiao and Jing-Ren Zhang, date: 08/05/2017, view: 6428, Q&A: 0

Streptococcus pneumoniae (pneumococcus) is an important human pathogen that causes pneumonia, meningitis, sepsis, and otitis media. This bacterium normally resides in the nasopharynx as a commensal, but sometimes disseminates to sterile sites of humans and causes local or systemic inflammation. This biphasic behavior of S. pneumoniae ...

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Digestion of Peptidoglycan and Analysis of Soluble Fragments Authors: Ryan E. Schaub and Joseph P. Dillard, date: 08/05/2017, view: 8668, Q&A: 0

Peptidoglycan (murein) is a vital component of the cell wall of nearly all bacteria, composed of sugars linked by short peptides. This protocol describes the purification of macromolecular peptidoglycan from cultured bacteria and the analysis of enzyme-digested peptidoglycan fragments using high performance liquid chromatography (HPLC). Digested ...

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A Protocol of Using White/Red Color Assay to Measure Amyloid-induced Oxidative Stress in Saccharomyces cerevisiae Authors: Vidhya Bharathi, Amandeep Girdhar and Basant K Patel, date: 08/05/2017, view: 7610, Q&A: 0

The yeast Saccharomyces cerevisiae (S. cerevisiae) harboring ade1 or ade2 mutations manifest red colony color phenotype on rich yeast medium YPD. In these mutants, intermediate metabolites of adenine biosynthesis pathway are accumulated. Accumulated intermediates, in the presence of reduced glutathione, are ...

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CRISPR/Cas9 Gene Editing in the Marine Diatom Phaeodactylum tricornutum Authors: Marianne Nymark, Amit Kumar Sharma, Marthe C. G. Hafskjold, Torfinn Sparstad, Atle M. Bones and Per Winge, date: 08/05/2017, view: 8398, Q&A: 0

The establishment of the CRISPR/Cas9 technology in diatoms (Hopes et al., 2016; Nymark et al., 2016) enables a simple, inexpensive and effective way of introducing targeted alterations in the genomic DNA of this highly important group of eukaryotic phytoplankton. Diatoms are of interest as model microorganisms in a variety of ...

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Molecular Biology

Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting Authors: Fuqiang Chen, Xiao Ding, Yongmei Feng, Timothy Seebeck, Yanfang Jiang and Gregory D Davis, date: 08/05/2017, view: 6329, Q&A: 0

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous ...

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Neuroscience

Extraction of Soluble and Insoluble Protein Fractions from Mouse Brains and Spinal Cords Author: Oliver Wirths, date: 08/05/2017, view: 18632, Q&A: 0

The current protocol details the preparation of soluble and insoluble protein lysates from mouse brain or spinal cord samples. In detail, tissue homogenization and sequential protein extraction are described. This procedure yields soluble and insoluble protein extracts that can be further processed in down-stream applications like ELISA or Western ...

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Preparation of Crude Synaptosomal Fractions from Mouse Brains and Spinal Cords Author: Oliver Wirths, date: 08/05/2017, view: 9524, Q&A: 0

The current protocol describes the preparation of crude synaptosomal fractions from mouse brain or spinal cord samples. In detail, a sequential protocol yielding crude synaptosomal and light membrane fractions is provided. This fast and easy method might be sufficient to assess the amount of synaptic proteins in down-steam applications like ...

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Plant Science

Polyamine and Paraquat Transport Assays in Arabidopsis Seedling and Callus Authors: Haoxi Chai, Yun Shen and Huazhong Shi, date: 08/05/2017, view: 4670, Q&A: 0

Polyamines (PAs) are polycationic compounds found in all living organisms and play crucial roles in growth and survival. We here show the ‘Polyamine and paraquat (PQ) transport assay’ protocol, which can be used to examine the uptake activity of PA/PQ transporters. We have used this protocol to demonstrate that PUT3 in Arabidopsis is a ...

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Overrepresentation Analyses of Differentially Expressed Genes in the Smut Fungus Ustilago bromivora during Saprophytic and in planta Growth Authors: Angelika Czedik-Eysenberg, Franziska Rabe, Heinz Ekker, Carmen Czepe and Armin Djamei, date: 08/05/2017, view: 5871, Q&A: 0

We have established the Ustilago bromivora–Brachypodium spp. interaction as a new model pathosystem for biotrophic fungal plant infections of the head smut type (Rabe et al., 2016). In this protocol, the methodology used for comparing gene expression between saprophytic and in planta growth of the fungus is ...

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Isolation of Cytosol, Microsome, Free Polysomes (FPs) and Membrane-bound Polysomes (MBPs) from Arabidopsis Seedlings Authors: Yonghui Zhao and Shengben Li, date: 08/05/2017, view: 7323, Q&A: 0

The plant endomembrane system plays vital roles for synthesis, modification and secretion of proteins and lipids. From the classic view, only mRNAs encoding secreted proteins could be targeted to the endoplasmic reticulum (ER) for translation via a co-translational translocation manner, however, recently this model has been challenged by ...

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Wheat Coleoptile Inoculation by Fusarium graminearum for Large-scale Phenotypic Analysis Authors: Lei-Jie Jia, Wan-Qiu Wang and Wei-Hua Tang, date: 08/05/2017, view: 7015, Q&A: 0

The ascomycete fungus Fusarium graminearum is a destructive fungal pathogen of wheat, barley and maize. Although reverse genetics and homologous recombination gene deletion methods have generated thousands of gene deletion mutants of F. graminearum, evaluating virulence of these fungal mutants is still a rate-limiting step. Here ...

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Isolation of Guard-cell Enriched Tissue for RNA Extraction Authors: Pirko Jalakas, Dmitry Yarmolinsky , Hannes Kollist and Mikael Brosche, date: 08/05/2017, view: 11364, Q&A: 0

This is a protocol for isolation of guard cell enriched samples from Arabidopsis thaliana plants for RNA extraction. Leaves are blended in ice-water and filtered through nylon mesh to obtain guard cell enriched fragments. With guard cell enriched samples, gene expression analysis can be done, e.g., comparing different gene ...

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Stem Cell

Differentiation of Human Induced Pluripotent Stem Cells (iPS Cells) and Embryonic Stem Cells (ES Cells) into Dendritic Cell (DC) Subsets Authors: Stephanie Sontag, Malrun Förster, Kristin Seré and Martin Zenke, date: 08/05/2017, view: 10199, Q&A: 0

Induced pluripotent stem cells (iPS cells) are engineered stem cells, which exhibit properties very similar to embryonic stem cells (ES cells; Takahashi and Yamanaka, 2016). Both iPS cells and ES cells have an extraordinary self-renewal capacity and can differentiate into all cell types of our body, including hematopoietic stem/progenitor cells ...

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Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants Authors: Alexandra S. Vagasi, Mohammad M. Rahman, Snehal N. Chaudhari and Edward T. Kipreos, date: 08/05/2017, view: 6072, Q&A: 0

The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. ...

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Mouse Müller Cell Isolation and Culture Authors: Xiao Liu, Luosheng Tang and Yongqing Liu, date: 08/05/2017, view: 7316, Q&A: 0

Müller cells are the major supportive and protective glial cells across the retina. Unlike in fish, they have lost the capacity to regenerate the retina in mammals. But, mammalian Müller cells still retain certain retinal stem cell properties with various degree of self-renewal and differentiation potentials, and thereby held a merit in cell-based ...

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